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Development of methodology for genome editing in Xenopus laevis using CRISPR/Cas9, targeting the rhodopsin… Feehan, Joanna Marie 2016

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	 DEVELOPMENT OF METHODOLOGY FOR GENOME EDITING IN XENOPUS LAEVIS USING CRISPR/CAS9, TARGETING THE RHODOPSIN GENE by Joanna Marie Feehan B.Sc.N., MacEwan University, 2013     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Cell & Developmental Biology)       THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2016 © Joanna Marie Feehan, 2016 	ii		ABSTRACT  Xenopus laevis is a commonly used research subject for retinal physiology and cell biology studies, but its utility is limited by the lack of a robust technology for generation of knock-out (KO) or knock-down (KD) phenotypes. However, new genome manipulation techniques involving CRISPR/Cas9 offer an opportunity for generating gene KOs in X. laevis. RNA-guided Cas9 endonuclease introduces double-stranded DNA breaks into the genome, which are either repaired by error-prone non-homologous-end joining (NHEJ), facilitating indel generation, or by less error-prone homology-directed repair (HDR), facilitating insertion of specific sequences. Rhodopsin was targeted for editing as the expected phenotypes, missing/malformed rod photoreceptor outer segments and lower rhodopsin content, are easily assayed. RNA and transgene methods for CRISPR/Cas9-mediated rhodopsin KOs and knock-ins (KI) in rod photoreceptors of X. laevis were tested, and an RNA injection protocol was developed and optimized. KOs were generated by in vitro transcription and microinjection of Cas9 mRNA, eGFP mRNA, and sgRNAs into in vitro fertilized eggs. Cas9 transgene cassettes were built and tested but editing attempts were unsuccessful. Indel mutations were identified by direct sequencing of PCR products and further characterized by sequencing individual clones. The extent of rhodopsin KO was quantified in 14 post-fertilization day-old tadpoles by anti-rod opsin dot blot assay of retinal extracts, and retinal phenotypes were assessed by cryosectioning and immunolabeling contralateral eyes for confocal microscopy. HDR-mediated KIs were generated by co-injection of a DNA repair fragment, with sufficient homology to the genomic region surrounding predicted dsDNA break-site. Heterologous expression of KIs was confirmed by immunohistochemistry. Delivery of Cas9 by RNA injection can produce high frequency homozygous and heterozygous KOs in X. laevis, permitting analysis in the first generation. I was able to obtain extensive KD generating very severe retinal degeneration phenotypes, and germline transmission of Cas9-mediated indels was confirmed. However, KO was never complete. Sequencing results indicate that first generation animals are chimeric containing 	iii		many independently derived indels. HDR-mediated KI techniques proved possible, but low in efficiency. These techniques significantly advance the utility of X. laevis as an experimental subject for cell biology and physiology studies.                      	iv		PREFACE All aspects of data collection and analysis were performed by myself. Design of research goals were in collaboration with my supervisor, Dr. Orson L. Moritz. Injection experiments were performed with the assistance of Beatrice Tam, Runxia Wen, Colette Chiu, and Paloma Stanar. Beatrice Tam was responsible for cryosectioning the eyes of JF851 and JF852 in section 3.1.  At the time of submission, no publications have arisen from work presented in this dissertation.  All procedures involving animals were approved by the UBC animal care committee under certificates A14-0275 Analysis of mechanisms underlying retinal degeneration 2014, and  A14-0274 In vivo analysis of mechanisms underlying retinal degeneration and photoreceptor structure 2014 issued to Orson L Moritz.               	v		TABLE OF CONTENTS ABSTRACT .................................................................................................................................. ii PREFACE ................................................................................................................................... iv TABLE OF CONTENTS …………………………………………………...……………………..…… v LIST OF FIGURES .................................................................................................................... viii LIST OF SYMBOLS AND ABBREVIATIONS ............................................................................. x ACKNOWLEDGEMENTS ........................................................................................................... xi 1. INTRODUCTION ...................................................................................................................... 1 1.1 Objective and hypothesis .......................................................................................... 1 1.2 Retinitis Pigmentosa ................................................................................................. 1 1.3 Rod photoreceptors and rhodopsin .......................................................................... 2 1.4 Xenopus laevis as genetic model ............................................................................. 2 1.6 Rationale ................................................................................................................... 5 1.7  Significance .............................................................................................................. 6 2. CRISPR/CAS9-MEDIATED INDEL MUTATIONS ................................................................... 7 2.1  Injected Cas9 mRNA is not toxic to in vitro fertilized embryos. ................................ 7 2.2  Injection of Cas9 mRNA with rhodopsin-targeting sgRNA rhosg3, or a combination of the rhodopsin-targeting sgRNAs rhosg1, rhosg2, and rhosg3, generates retinal degeneration phenotypes. ........................................................................................ 8 2.3 Co-injection of eGFP mRNA enriches for more severe retinal degeneration phenotype, but raising at room temperature for first 24 hours does not. ................ 10 2.4 Neither injecting recombinant Cas9 nor raising at 16°C for first 24 hrs provokes more severe retinal degeneration phenotype than Cas9 mRNA or raising at 18°C.12 2.5 Increasing amount of RNA injected generates more deleterious retinal degeneration phenotype. .............................................................................................................. 14 	vi		2.6 Two rhodopsin homeologs exist in Xenopus laevis, and two alleles have been identified for each homeolog. .................................................................................. 16 2.7 The rhosg1 targets all homeologs generating more severe retinal degeneration phenotype. .............................................................................................................. 19 2.8 Germline transmission of edited rhodopsin gene. .................................................. 26 2.9 The rhosg4 targets the last exon of rhodopsin inducing retinal degeneration. ....... 28 2.10 Outer segments of rhodopsin knock-out rod photoreceptors are malformed. ........ 29 2.11 Mislocalized rhodopsin proteins likely indicate in-frame mutations. ....................... 30 3. CRISPR/CAS9-MEDIATED INSERTION OF EXOGENOUS DNA ........................................ 31 3.1 Heterologous expression of rhodopsin-GFP fusion protein through injection of rhosg4 with repair template containing eGFP sequence and rhodopsin homology. 31 3.2 Heterologous expression of rhodopsin with novel M13F epitope through injection of rhosg1 with repair template containing rhodopsin homology with a missense mutation. ................................................................................................................. 33 4. RNA-GUIDED CAS9 GENE DRIVE SYSTEM ....................................................................... 35 4.1 Confirming enzymatic activity of Cas9-GFP and Cas9-FLAG by mRNA injection. . 35 4.2 A rod opsin-promoter driven Cas9-GFP, and an X. tropicalis U6 promoter-driven sgRNA. ................................................................................................................... 37 4.3 A CMV-promoter driven Cas9-GFP, and human U6 promoter-driven sgRNA. ....... 38 5. METHODS .............................................................................................................................. 40 5.1 Cas9 and eGFP mRNA In Vitro Transcription ........................................................ 40 5.2 Cloning sgRNA target-site sequences into sgRNA backbone of pDR274 .............. 41 5.3 In Vitro Transcription of sgRNA .............................................................................. 41 5.4 RNA Microinjection ................................................................................................. 42 5.5 HDR Specific Methods ............................................................................................ 44 5.6 Transgenesis Specific Methods .............................................................................. 45 	vii		CONCLUSION ........................................................................................................................... 47 BIBLIOGRAPHY ........................................................................................................................ 49 APPENDICES ............................................................................................................................ 56 APPENDIX A. Sequences for each allele target-site with respective primer sequences for amplification. ................................................................................................ 56 APPENDIX B. Cloning sgRNAs into pDR274 for downstream in vitro transcription ............... 57 APPENDIX C. Full Protocol for RNA Injection Experiments ................................................... 58 APPENDIX D. Excision of RHO by co-injection of rhosg3 and rhosg4 .................................. 62 APPENDIX E. eGFP-HDR Construct Design ......................................................................... 63 APPENDIX F. M13F-HDR Construct Design ......................................................................... 64 APPENDIX G. Cloning methods for sgRNAs into hU6 & xtU6 (Ligation & Gibson Assmbly) . 65 APPENDIX H. XOP-Cas9-GFP-xtU6 ..................................................................................... 68 APPENDIX I. CMV-Cas9-GFP-hU6 ....................................................................................... 70 APPENDIX J. Example of direct sequencing base call analysis. ........................................... 72 APPENDIX K. WT Alignments for section 2.7 ........................................................................ 74 APPENDIX L. NLS Change in Cas9-GFP .............................................................................. 76 APPENDIX M. xtU6 design error ............................................................................................ 77       	viii		LIST OF FIGURES FIGURE 2.A  Survival of embryos injected with increasing amounts of Cas9 mRNA and 700pg of eGFP mRNA. The number of GFP-positive embryos were recorded for each group daily. ............................................................................................................ 8 FIGURE 2.B Injection of 6ng Cas9 mRNA with 200pg of rhodopsin-targeting sgRNA rhosg3, or a combination of the rhodopsin-targeting sgRNAs rhosg1, rhosg2, and rhosg3, generates retinal degeneration phenotypes. . ..................................................... 10 FIGURE 2.C Co-injection of eGFP mRNA enriches for more severe retinal degeneration phenotype, but raising at room temperature for first 24 hours does not. ............ 12 FIGURE 2.D Neither injecting recombinant Cas9 nor raising at 16˚c for first 24hrs provokes more severe retinal degeneration phenotype than Cas9 mRNA or raising at 18˚c. ............................................................................................................................ 14 FIGURE 2.E Increasing the amount of Cas9 mRNA and rhosg3 injected generates a more severe retinal degeneration phenotype. . ............................................................ 15 FIGURE 2.F Alignments of identified rhodopsin alleles. . ........................................................ 18 FIGURE 2.G rhosg1 targets all homeologs generating more severe retinal degeneration phenotype. .......................................................................................................... 24 FIGURE 2.H rhosg1 targets all homeologs generating indels in injected embryos within 24 hours of fertilization. ............................................................................................ 25 FIGURE 2.I  Germline transmission of edited rhodopsin gene. . ............................................. 28 FIGURE 2.J rhosg4 targets the last exon of rhodopsin inducing retinal degeneration. .......... 29 FIGURE 2.K Observations made on morphology. ................................................................... 30  FIGURE 3.A Heterologous expression of rhodopsin-GFP fusion protein through injection of rhosg4 with repair template containing eGFP sequence and rhodopsin homology. ............................................................................................................................ 32 	ix		FIGURE 3.B Heterologous expression of rhodopsin with novel M13F epitope through injection of rhosg1 with repair template containing rhodopsin homology with point mutation. ............................................................................................................. 34  FIGURE 4.A Confirming enzymatic activity of Cas9-GFP, Cas9-FLAG by mRNA injection. ... 36 FIGURE 4.B  A rod opsin-promoter (XOP) driven Cas9-GFP, and an X. tropicalis U6 promoter-driven sgRNA. ..................................................................................................... 38 FIGURE 4.C A CMV-promoter driven Cas9-GFP, and human U6 promoter-driven sgRNA. ... 39                   	x		LIST OF SYMBOLS AND ABBREVIATIONS Cas  – CRISPR-associated CRISPR – Clustered regularly interspaced short palindromic repeats dpf  – Day-post-fertilization DSB  – double-stranded DNA break dsDNA  – double-stranded DNA HDR  – Homology directed repair IN  – Inner Segment INL  – Inner Nuclear Layer KO  – Knock-out KI  – Knock-in KD  – Knock-down NHEJ  – Non-homologous end joining ONL  – Outer Nuclear Layer OS  – Outer segment RD  – Retinal Degeneration rhosg1  – rhodopsin-targeting sgRNA, targets all homeologs in the first exon rhosg3  – rhodopsin-targeting sgRNA, targets one allele of each homeologs exon 1 rhosg4  – rhodopsin-targeting sgRNA, targets all homeologs in last exon RP  – Retinitis Pigmentosa RPE  – Retinal Pigment Epithelium sgRNA  –  single-guide RNA ssDNA  – single-stranded DNA TALEN – Transcription activator-like effector nuclease  ZFN  – Zinc-finger nuclease  	xi		ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my advisor Prof. Orson L. Moritz for his continuous support of my MSc study and research, and for his patience, motivation, guidance, and knowledge. I am grateful for his dedication to my project, and for the stimulating discussions regarding my thesis work and general science.  Besides my advisor, I would like to thank the rest of my thesis committee: Prof. Robert S. Molday and Kurt Haas for their insightful comments and encouragement. I thank Beatrice Tam for her guidance and support when performing laboratory skills and for her mentorship when learning how to perform the microinjection experiments. I thank Colette Chiu for her assistance with maintaining my animals, for preparing the WT animals, and for her assistance with the microinjection experiments. I thank Runxia Wen for her support and encouragement, and for her assistance with the microinjection experiments. I thank Paloma for her assistance with microinjection experiments and for providing constructive feedback while learning the protocols I have developed. Finally, I would like to thank my uncle and aunt Chris and Shauna Somerville for their scientific guidance and support, my parents Anna Somerville and Mark Feehan, the rest of my family, and my friends for their encouragement.  	1		1. INTRODUCTION 1.1 Objective and hypothesis The overall objective of my thesis project was to elucidate the necessary requirements for CRISPR/Cas9 genome editing in Xenopus laevis using Rhodopsin, a gene that we study for its role in multiple retinal genetic disorders, as the initial test target-site. We hypothesized that using CRISPR/Cas9 we would be able to generate random insertions or deletions (indels) of DNA in the X. laevis rhodopsin gene. We further hypothesized that in the presence of homologous DNA, CRISPR/Cas9 would integrate exogenous sequence into the rhodopsin gene of X. laevis. Rhodopsin is a useful initial test subject as it is easily assayed by histology and immunochemistry, and it is the subject of ongoing investigations in our lab related to the mechanisms of retinal degeneration (RD).   1.2 Retinitis Pigmentosa  Retinitis Pigmentosa (RP), an inherited RD disorder of rod photoreceptors1, occurs in approximately 1 in 4000 individuals2. RP is considered one of the most genetically heterogeneous disorders in humans3-6 as more than 50 genes and hundreds of mutations have been implicated7-10. Moreover, the same mutation in different individuals, even within the same family, may present with distinctly different symptoms3. Most cases of RP cause primary degeneration of rod photoreceptors and secondary degeneration of cones, therefore, patients usually present with night blindness and peripheral vision loss, and in later life, central vision impairment11. In severe cases, symptoms may proceed to complete blindness6,12. There are neither cures nor effective treatments for RP currently available13,14. The most common mutations associated with RP are those that result in misfolded rhodopsin protein1,3,6,15. However, the role of rhodopsin in RP is not fully understood.   	2		1.3 Rod photoreceptors and rhodopsin The light-sensitive rod and cone photoreceptors are contained in the outer layer of the neural retina. The rod outer segment (OS) is composed of densely packed membrane disks, each disk containing high concentrations of tightly packed rhodopsin photopigments1. The OS contains ten million to one billion rhodopsin molecules, depending on the number of disks and their diameter16. Rhodopsin is composed of a rod opsin covalently linked to an 11-cis-retinal chromophore through Lys2965,9,10,17. When the 11-cis-retinal absorbs a photon of light, it is isomerized to all-trans-retinal inducing a conformational change that transiently activates the opsin17. Hundreds of guanine nucleotide-binding (G) protein molecules interact with a single light-activated rhodopsin, initiating downstream signaling of second-messenger pathways17,18.  Researchers are only beginning to understand the role of rhodopsin in photoreceptor membrane trafficking and OS biogenesis19. However, it is known that rhodopsin is essential for disk formation, as the rods of homozygous rhodopsin knock-out (KO) mice either do not form elaborated rod OSs16, or OSs are absent20. Disk enlargement in rods of transgenic mice that overexpress rhodopsin suggests that disk size is dependent upon the amount of rhodopsin transported from inner segment16. Furthermore, Makino et al. found that smaller disks were formed in hemizygous rhodopsin KO mice that expressed half the normal amount of rhodopsin16.    1.4 Xenopus laevis as genetic model Although several animal genetic models exist for RP studies20,21,22-24, researchers have yet to elucidate the pathways leading from mutation to rod death25. X. laevis offers a unique model in photoreceptor studies as the volume of OS membrane trafficking is much higher in amphibians than in mammals26, therefore, biochemical and morphological data can be correlated in a single experimental animal19. Initially, X. laevis served as a developmental and embryogenesis model for many discoveries of fundamental mechanisms of vertebrate cell 	3		biology27,28. X. laevis was thought to be an undesirable genetic model as it’s genome is allotetraploid, hypothesized to be the hybridization of two species29 or a duplication event during evolution30, making specific gene targeting difficult31. However, the Xenopus Genome Initiative greatly increased the function of X. laevis as a genetic model organism32. In fact, the evolutionary distance from mammals to amphibians proved ideal for bioinformatic comparison28. With a wide range of material such as nuclei33, nucleic acids28, and proteins28 easily injected into eggs and embryos, X. laevis has served as one of the best models for testing the function of genetic products30. Furthermore, X. laevis often produces thousands of embryos at a time that can be harvested for versatile analysis applications28,34. Accordingly, in the last twenty years methods for genetic manipulation of X. laevis have become standard practice35. The genome of X. laevis is not complete, but frequent updates of the online database, XenBase, indicate that sequencing of the genome is ongoing and likely to be completed soon.   1.5 Methods for site-specific genome editing Available genomic-editing tools for KO and KD include transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), and CRISPR/Cas9. These tools generate double-stranded DNA breaks (DSBs), wherein endogenous repair mechanisms provide opportunities for genomic editing. There are two repair mechanisms for DSB, non-homologous end joining (NHEJ) and homology-directed repair (HDR)36. NHEJ is an error-prone repair system that will either insert or delete random nucleotides at the break-site, potentiating premature stop codons that may render the transcript nonfunctional37. In the presence of template DNA with sufficient homology around the predicted cut-site, HDR will integrate a foreign sequence38. ZFNs are fusions of a non-specific endonuclease domain, FokI, with zinc-finger DNA-binding domains39. Several limitations prevent wider application including limited modularity40, generation of off-target cleavage41,42, high expense, and the need for extensive optimization and binding-specificity screening43. TALENs, similar to ZFNs, are composed of TAL 	4		effector DNA-binding domains fused to FokI nuclease domains39. TALEN utility is limited by specificity requirements and a costly, labor intensive construction38. Furthermore, analyses have confirmed that TALENs can still show indel mutations at detectable frequencies by up to nine mismatches44. An alternate technology, RNA interference, showed initial promise as a KD strategy in X. laevis45, but has since been widely acknowledged as ineffective46.   CRISPR/Cas9 is based on an adaptive immune system identified in bacteria and archaea. The RNA-mediated Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a defense response to invading nucleic acids47. The host integrates fragments of foreign nucleic acid as an array of genetic records into a CRISPR loci. These fragments are then transcribed as CRISPR-derived RNAs (crRNAs)48 for detection and destruction of recurrent invaders47,48. Included in this operon are CRISPR-associated (Cas) genes that when expressed mediate defense mechanisms47. Immunity is achieved as a three-phase system: the adaptive phase, wherein the complementary sequence is recorded, followed by expression and interference phases mediating transcription and target recognition by crRNAs47. There are three types of CRISPR/Cas systems, however, current biotechnology applications are based on type II47. In type II, Cas9 cleavage activity depends on the base-pair structure formed between a trans-activating crRNA (tracrRNA) and the target recognition crRNA47. Cleavage specificity is determined by the complementarity of this base-pair structure to target-site, and juxtaposition of target-site next to a protospacer adjacent motif (PAM) in the target nucleic acid47. Jinek et al.47 were the first to demonstrate that a chimeric single-guide RNA (sgRNA) combining critical sequences of both crRNA and tracrRNA, can be programmed to target specific DNA sites, and in doing so have offered a versatile and powerful new tool for genetic manipulation in virtually any model organism.  	5		1.6 Rationale CRISPR/Cas9 is unique in contrast to ZFN and TALEN technologies, as the utilization of the sgRNA increases specificity and efficiency49; a wide variety of targeted genome engineering applications have been successful38 in multiple different model systems such as Drosophila50, zebrafish51-55, and mice54. Most significant to this thesis work, CRISPR/Cas9 has been successfully applied in the close relative of X. laevis, Xenopus tropicalis56-59. Blitz et al. (2013) utilized NHEJ by targeting the tyrosinase gene, resulting in albino X. tropicalis, and reported a success rate of 84% after sequencing 19 independent clones from a single injected embryo57. Recently, Jaffe et al. showed successful integration of a fluorescent reporter for editing with Cas9 injection and co-injection of a homology repair template in X. laevis multiciliated cells60. Successful HDR attempts in other models have been reported, with eukaryotes requiring repair fragment (RF) homology to be greater than 240bp61, and Drosophila, by embryo injection, higher frequency rates with increased with homology lengths greater than 1kb62. However, Cas9 technology is still in its infancy, Jinek et al. proposed the single sgRNA in 201247, and the degree of specificity in bioapplications of this system have not been fully elucidated. Jinek et al. reported that mismatched sequences can still be cleaved, making the likelihood of off-target effects a considerable concern47. Therefore, application of Cas9 must include comprehensive analyses for potential off-target effects49. With stringent planning, controls, and analysis, CRISPR/Cas9 has the potential to be an excellent technique for targeted genomic editing in X. laevis. For development of CRISPR/Cas9 methodology in X. laevis, rhodopsin has several properties that make it an ideal initial test target. Rhodopsin is expressed at very high levels exclusively in rod photoreceptors, therefore, KO of rhodopsin would not likely be lethal and quantification methods can be sensitive. There are numerous high affinity anti-rhodopsin antibodies available, as well as established immunoassay protocols in our laboratory, to assay 	6		for RD phenotypes associated with missing and malformed rod OS, as identified in rhodopsin KO mice.   1.7  Significance X. laevis is a useful model for retinal studies19,26,28, and although gain-of-function transgenic X. laevis are generated relatively easily to model dominant disorders63,64, researchers have previously been unable to engineer loss-of-function mutations28 such as KO and knock-down (KD) for recessive modeling. Knock-in (KI) transgenesis techniques by restriction enzyme mediated integration (REMI) are easy and efficient in X. laevis65 and have become standard practice35. Gene KO would be an extremely useful complement to KI transgenesis techniques for mechanistic inquiries, and would allow modeling of recessive forms of inherited retinal disorders. Furthermore, the outcome of this research would also generate a rhodopsin KO line of X. laevis, a valuable tool for cell biology studies and disease modeling.                   	7		2. CRISPR/CAS9-MEDIATED INDEL MUTATIONS  2.1  Injected Cas9 mRNA is not toxic to in vitro fertilized embryos. The goal of my project was to elucidate the necessary requirements for application of CRISPR/Cas9 technology in X. laevis. We based the preliminary experiments on the paper by Grainger et al.59, wherein they generated tyrosinase KOs in X. tropicalis, a close relative of X. laevis, using Cas9 mRNA injection. The author’s recommended microinjection dose was 300-2000pg of Cas9 mRNA and 50-200pg sgRNA per embryo59. Due to size differences between X. laevis and X. tropicalis oocytes, 1-1.3mm vs. 0.7-0.8mm in diameter respectively, we hypothesized that an increased dosage of injected RNA would not be toxic to X. laevis embryos. We, therefore, compared the survival rates of in vitro fertilized embryos injected with 14ng, 5ng, 1.5ng, 0.5ng, or 0.15ng of Cas9 mRNA at the single-cell stage. 700pg of eGFP mRNA was co-injected as a reporter for successful Cas9 mRNA delivery. Cas9 and eGFP mRNA were in vitro transcribed using Ambion’s T7 mMessage mMachine Ultra kit for assembly of capped and poly-adenylated mRNA transcripts. Linear pMLM3613 (Addgene), encoding Cas9 on a T7 promoter, was used as template for Cas9 mRNA synthesis. A detailed protocol for both in vitro transcription and microinjection can be found in Appendix C.  Injected embryos were screened 1-day post-fertilization (dpf) for GFP fluorescence. GFP-positive (GFP+) embryos were collected for further analysis. Wild-type (WT) in vitro fertilized embryos were also collected and subjectively compared, however, as they were not injected with eGFP mRNA, the survival rates were not included in these results. On Day 5, the embryos injected with 0.15ng, 0.5ng, or 1.5ng of Cas9 mRNA were destroyed as the survival rates between these and the higher dosages were not different (Fig. 2.A). The GFP+ embryos injected with 14ng or 5ng were raised to 14-dpf (stage 48), the standard time-point for sacrifice and analysis in our lab. Toxicity was not evident in any of the injection groups, as survival rates remained constant after 2-dpf (Fig. 2.A). At 14-dpf, morphology of tadpoles derived from 	8		injected embryos was not different than morphology of tadpoles derived from WT uninjected embryos (data not shown).  Figure 2.A  Survival of embryos injected with increasing amounts of Cas9 mRNA and 700pg of eGFP mRNA. The number of GFP-positive embryos were recorded for each group daily.   2.2  Injection of Cas9 mRNA with rhodopsin-targeting sgRNA rhosg3, or a combination of the rhodopsin-targeting sgRNAs rhosg1, rhosg2, and rhosg3, generates retinal degeneration phenotypes. As injection of 14ng Cas9 mRNA and 700pg eGFP mRNA into in vitro fertilized single-cell embryos was not toxic and did not produce dysmorphic tadpoles, three rhodopsin-targeting sgRNAs were designed using ZiFit,: rhosg3, in the first exon of rhodopsin adjacent to the start codon, rhosg2, downstream-adjacent to rhosg3, and rhosg1, approximately 100bp downstream of rhosg3 (Appendix A). The published mRNA sequence for X. laevis rhodopsin was used for sgRNA design66. Corresponding oligomers were synthesized and cloned into linear pDR274 (Addgene). A detailed ligation-cloning protocol can be found in Appendix B. Cloning success was confirmed by sequencing, and positive clones were linearized and used as template DNA for in vitro transcription reactions with the Ambion MAXIscript kit. A more detailed protocol can be found in Appendix C.  	9		As previously stated in chapter 2.1, Grainger et al. recommended an injection dose of 50-200pg sgRNA. Based on the disparate sizes of X. laevis (1-1.3mm diameter) and X. tropicalis oocytes (0.7-0.8mm diameter), we hypothesized that the X. laevis embryos may require more sgRNA for editing and would be able to tolerate larger quantities and injection volumes without toxicity concerns. Single-cell in vitro fertilized embryos were injected with 6ng Cas9 mRNA, 700pg eGFP mRNA, and 200pg of either rhosg1, rhosg2, rhosg3, or a combined total dose of 200pg or 400pg. Tadpoles were raised in 0.1XMMR to 14-dpf at which point they were sacrificed by pithing in accordance with animal care protocols. One eye was collected for isolation of retinal extracts by solubilization, the contralateral eye was fixed in paraformaldehyde for immunohistochemistry analysis, and genomic DNA (gDNA) was extracted from the tail tissue. Analysis of retinal extracts by dot blot indicated that tadpoles from embryos injected with rhosg3 had significantly lower levels of rod opsin relative to tadpoles from uninjected WT control embryos, and there was no difference in measured rod opsin value between retinal extracts of tadpoles from embryos that were injected with rhosg3 or combined amounts of rhosg1, rhosg2, and rhosg3 (Fig. 2.B.a). Contralateral eyes of the tadpoles used in the dot blot were cryosectioned and immunolabeled for analysis for RD phenotypes. A similar level of RD was observed in tadpoles that arose from embryos injected with rhosg3 and combined rhosgRNAs (Fig. 2.B.b). RD was not observed in tadpoles from embryos injected with rhosg1 or rhosg2 (data not shown). Based on the comparable performance of rhosg3 to injection of the combined rhosgRNAs, we chose to utilize rhosg3 as a single sgRNA in further experiments.  	10		 Figure 2.B Injection of 6ng Cas9 mRNA with 200pg of rhodopsin-targeting sgRNA rhosg3, or a combination of the rhodopsin-targeting sgRNAs rhosg1, rhosg2, and rhosg3, generates retinal degeneration phenotypes. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by ANOVA showed a statistically significant difference between groups (P = 3.40 X 10-5).  Individual comparisons were subsequently made using Tukey test (P values shown on graph). The mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos injected with rhosg3 + Cas9 mRNA, 200pg of rhosg1, rhosg2, and rhosg3 combined + Cas9 mRNA, and 400pg of rhosg1, rhosg2, and rhosg3 + Cas9 mRNA were significantly different from the mean total rod opsin values of the uninjected WT. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The left panels show signals from all channels in order to assess rod outer segment morphology: Wheat germ agglutinin (WGA) was used to label membranes in red, B630N was used to label rhodopsin in green, and DAPI was used to label nuclei in blue. The right panels show rhodopsin localization to rod outer segments in green. The retinal layers are labeled as the: Inner Nuclear Layer (INL) containing horizontal and bipolar cell bodies, Outer Nuclear Layer (ONL) containing rod and cone photoreceptor cell bodies, Inner Segment (IS) of photoreceptors, Outer Segment (OS) of photoreceptors, and Retinal Pigment Epithelium (RPE). The tadpole retinas from embryos injected with rhosg3 and 400pg rhosg1, rhosg2, and rhosg3 combined showed a similar level of retinal degeneration.   2.3 Co-injection of eGFP mRNA enriches for more severe retinal degeneration phenotype, but raising at room temperature for first 24 hours does not. Delivery of Cas9 mRNA and rhosg3 generated RD phenotypes in tadpoles from injected embryos, however, the severity was less than anticipated. The endogenous CRISPR/Cas9 	11		system exists in bacteria48 with 37°C as the optimal growth temperature. Thus, we investigated whether raising the ambient temperature would result in more severe RD phenotypes. Khokha et al. report that X. laevis can develop well when raised anywhere between 16°C and 25°C67. We compared the RD phenotypes of tadpoles from embryos incubated at 18°C or room temperature (“RT”, ~22°C) for 24 hours after RNA injection. We also attempted to enrich the collection of tadpoles analyzed for RD phenotypes by co-injection of embryos with eGFP mRNA as a reporter for sufficient Cas9 mRNA and rhosgRNA delivery, eliminating those from downstream analysis that were accidentally uninjected, not injected due to a plugged needle, or in which the injected solution leaked out of the embryo. We suspected that embryos with less intense GFP-fluorescence did not receive a full dose of RNA, and therefore, we compared the RD phenotypes of tadpoles from embryos with high-intensity GFP-fluorescence vs. low intensity. Single-cell in vitro fertilized embryos were injected with 6ng Cas9 mRNA, 700pg eGFP mRNA, and 2ng rhosg3. Half were allowed to develop at 18°C while the other half were placed at RT for 24 hours after which they were transferred to 18°C indefinitely. One-dpf embryos from both incubation temperatures were visually inspected for GFP-fluorescence intensity, and separated into four analysis groups for comparison: (1) high GFP 18°C, (2) low GFP 18°C, (3) high GFP RT, and (4) low GFP RT. GFP-negative (GFP-) embryos were not analyzed. Uninjected in vitro fertilized WT embryos raised at 18°C served as the controls. Most embryos that were allowed to develop at RT exhibited morphological abnormalities and did not result in viable tadpoles (data not shown). Only those tadpoles that developed with normal morphology were analyzed. The retinal extracts of tadpoles from the “high GFP 18°C” and “high GFP RT” groups had significantly lower levels of rod opsin versus the WT controls (Fig. 2.C.a). There was no significant difference in rod opsin values at between “high GFP 18°C” and “high GFP RT”. None of the embryos from “low GFP RT” developed into viable tadpoles. Therefore, in further experiments we co-injected eGFP mRNA, incubated injected embryos at 	12		18°C, and performed analyses on tadpoles from embryos with higher intensity GFP-fluorescence.   Figure 2.C Co-injection of eGFP mRNA enriches for more severe retinal degeneration phenotype, but raising at room temperature for first 24 hours does not. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by ANOVA showed a statistically significant difference between groups (P = 9.60 X 10-6).  Individual comparisons were subsequently made using Tukey test (P values shown on graph). The mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos with high intensity GFP fluorescence 1-dpf were significantly different from the mean total rod opsin values of the uninjected WT and low intensity GFP fluorescence 1-dpf. There was no statistically significant difference between the mean total rod opsin values of embryos raised at 18°C versus RT. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The tadpole retina from an embryo with high intensity GFP fluorescence at 1-dpf raised at 18°C is degenerated.    2.4 Neither injecting recombinant Cas9 nor raising at 16°C for first 24 hrs provokes more severe retinal degeneration phenotype than Cas9 mRNA or raising at 18°C. Injection of Cas9 mRNA necessitates a delay of an unknown amount of time before sufficient Cas9 to mediate editing will be translated. Therefore, we considered the possibilities that slowing cell division by reducing the ambient temperature might promote greater levels of editing, or that injection of Cas9 protein would promote greater levels of editing. We tested 	13		recombinant Cas9 from NEB for editing at the single-celled stage. This was attempted with in vitro fertilized embryos and embryos fertilized by injected sperm nuclei, raised at 18°C or incubated at 16°C for the first 24hrs before transfer to 18°C. The sperm nuclei were either incubated prior to injection with recombinant Cas9 enzyme and sgRNA, or there was no incubation with nuclei prior to injection. Uninjected In vitro fertilized WT embryos, raised at 18°C or incubated at 16°C for the first 24hrs, served as controls.  In the first method of fertilization by nuclei injection, recombinant Cas9 and rhosg3 were incubated at room temperature for 10 minutes and then placed on ice. They were then incubated with sperm nuclei for 5 minutes, egg extract for 10 minutes (to decondense sperm nuclei chromatin), and then injected into unfertilized embryos. In the second method of fertilization by nuclei injection, recombinant Cas9 and rhosg3 were again incubated at room temperature for 10 minutes and then placed on ice. However, in this method sperm nuclei were incubated with only the egg extract and not the Cas9-rhosg3 complex prior to injection. Finally, the Cas9-rhosg3 complex was injected into in vitro fertilized embryos as a third method.  There were no significant differences between rod opsin values of WT retinal extracts and any of the respective temperature injection groups. The available recombinant Cas9 enzyme used in this experiment did not have a nuclear localization signal (NLS). Potentially, the resultant low density of Cas9 localized to the nucleus accounts for the lack of retinal degeneration observed in tadpoles from embryos injected with recombinant Cas9. A subsequent experiment was attempted with recombinant Cas9 encoding an NLS, however, the needles became plugged immediately after loading the injectable and data could not be collected. 	14		 Figure 2.D Neither injecting recombinant Cas9 nor raising at 16˚C for first 24hrs provokes more severe retinal degeneration phenotype than Cas9 mRNA or raising at 18˚C. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles raised at 16˚C, for first 24hrs before transfer to 18˚C, quantified by anti-rod opsin dot blot assay. There were no statistically significant differences between groups. (b) Total rod opsin of retinal extracts from 14-dpf tadpoles raised at 18˚C quantified by anti-rod opsin dot blot assay. There were no statistically significant differences between groups.    2.5 Increasing amount of RNA injected generates more deleterious retinal degeneration phenotype. While our methods of raising at 18°C and co-injecting eGFP mRNA proved optimal, the KO phenotypes were still not complete, as many rods expressing rhodopsin were still present in sectioned retinas (Fig 2.C.b). We hypothesized that by injecting an increased amount of Cas9 mRNA and/or sgRNA, a more severe RD phenotype would be observed. We injected single-cell in vitro fertilized embryos with either 6ng Cas9 mRNA and 400pg rhosg3, 6ng Cas9 mRNA and 2ng rhosg3, or 30ng Cas9 mRNA and 2ng rhosg3. All embryos were co-injected with 700pg eGFP mRNA and screened for GFP-fluorescence. The concentration of RNA after in vitro transcription was increased by ethanol precipitation (Appendix C).  All injection groups had significantly lower values of rod opsin versus the uninjected WT controls. There were significantly lower values of rod opsin from retinal extracts of tadpoles that 	15		arose from embryos injected with 30ng Cas9 mRNA and 2ng rhosg3, versus tadpoles that arose from embryos injected with 6ng Cas9 mRNA and 400pg Cas9 mRNA. However, the contralateral cryosectioned and immunolabeled eyes of tadpoles from embryos injected with 30ng Cas9 mRNA and 2ng rhosg3 did not exhibit a drastically more severe RD phenotype than those eyes of tadpoles from embryos injected with 6ng Cas9 mRNA and 400pg rhosg3.  Figure 2.E Increasing the amount of Cas9 mRNA and rhosg3 injected generates a more severe retinal degeneration phenotype. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by ANOVA showed a statistically significant difference between groups (P = 4.12 X 10-19).  Individual comparisons were subsequently made using Tukey test (P values shown on graph). The mean total rod opsin values from retinal extracts of whole tadpole eyes from all groups of embryos injected with varying amounts of Cas9 and rhosg3 were significantly different from the mean total rod opsin values of the uninjected WT. There was a statistically significant difference in the mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos injected with 6ng Cas9 + 400pg rhosg3 and 30ng Cas9 + 2ng rhosg3. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The tadpole retinas from embryos injected with 6ng Cas9 mRNA + 2ng rhosg3 and 30ng Cas9 mRNA + 2ng rhosg3 showed a similar level of retinal degeneration.   	16		2.6 Two rhodopsin homeologs exist in Xenopus laevis, and two alleles have been identified for each homeolog. X. laevis is an allotetraploid species, hypothesized to have undergone a whole-genome duplication after the hybridization of two closely related diploid species68. Matsuda et al. have recently identified the presence of “long” and “short” homeologous chromosome pairs in the X. laevis karyotype, and have proposed a nomenclature that refers to chromosome sets as XLA1L and XLA1S, XLA2L and XLA2S, and so on for the 9 sets68.  As the genome of X. laevis is not complete, but nearing completion, initial rhodopsin-targeting sgRNAs were designed based on the published rhodopsin mRNA sequence66. However, sequencing analyses revealed the presence of multiple rhodopsin sequences in the animals used in my experiments. I then discovered that the published rhodopsin coding DNA sequence (cds)69 available on Xenbase was not identical to the published mRNA sequence found on the National Centre for Biotechnology Information (NCBI). Using BLAST searches, and alignments to the published RHO cds and available mRNA accessions on NCBI, I identified two distinct rhodopsin genes in Xenbase, one located on chromosome XLA4L (Rho4L) and the other on XLA4S (Rho4S). These sequences have not been identified as rhodopsin on Xenbase and neither has the published RHO cds been given a genetic locus on Xenbase. These genes are most likely homeologs (paralogs arising from polyploidy). I have identified two alleles distinct to each homeologous gene, Rho4L1-1, Rho4L1-2 for the Rho4L gene and Rho4S1-1, Rho4S1-2 for the Rho4S gene. I acknowledge that there are potentially more unidentified Rho4S and Rho4L alleles in X. laevis, as it is generally used as an outbred animal model, although inbred lines, such as the J-strain utilized in the genome sequencing project, have been established. rhosg3 and rhosg2 target Rho4S1-2 and Rho4L1-2 while rhosg1 is complementary to all identified alleles of both homeologs and was thus implemented in further experiments for generation of full KO phenotypes (Fig. 2.F.a). The PAM associated with rhosg2 is abolished in Rho4L1-1 and Rho4S1-1. Distinct primers were designed 	17		for amplification of Rho4L and Rho4S (Appendix A). The Rho4S1-2 allele was identified late in my thesis research through what appears to be a fortuitous mispriming event. This allele does not seem to exist as an available reference sequence online, therefore, the full sequence of this allele remains unknown (Fig. 2.F.a).  The peptide alignment in Fig. 2.F.b shows locations where residues are variable between alleles. In certain positions, the Rho4S alleles encode the same amino acid and the Rho4L alleles code for the same peptide that is different from the Rho4S. In other positions, the Rho4S1-2 and the Rho4L1-2 encode the same peptide, while the Rho4S1-1 and Rho4L1-1 encode for a different amino acid from the 1-2 alleles. There are also positions wherein one allele has a unique amino acid substitution from the others. This data indicates that all alleles are distinct, and encode distinct amino acid sequences that are highly similar. Furthermore, the amino acid substitutions do not appear to be deleterious. It would be interesting to determine whether all homeolog copies encode a functional rhodopsin. However, as known critical residues (highlighted in blue in Fig. 2.F.b and Fig. 2.F.c) are not substituted, it seems likely that both homeologs encode functional rhodopsins.   	18		 Figure 2.F Alignments of identified rhodopsin alleles. (a) Coding DNA sequence alignment of Rhosg4L1-1, Rho4L1-2, Rho4S1-1, Rho4S1-2. rhosg1 target-site sequence alignment in yellow, rhosg2 target-site sequence alignment in green, rhosg3 target-site sequence alignment in red, and rhosg4 target-site sequence alignment in black. rhosg3 is perfectly complementary to Rho4L1-2 and Rho4S1-2 while there are mismatches to Rho4L1-1 and Rho4S1-1 at positions 14 and 20 distal to the PAM. rhosg2 only targets Rho4L1-1 and Rho4S1-1 as the PAM site is abolished by nucleotide changes in Rho4L1-2 and Rho4S1-2. 	19		rhosg1 and rhosg4 target both alleles for Rho4L and Rho4S. (b) Peptide sequence alignment from translated nucleic acid sequence. (c) Two-dimensional model of bovine rhodopsin70. Highlighted in pink are some of the residues that correspond with known RP mutations. Important residues necessary for WT activity are highlighted in blue: Glu-113, the counter-ion to the PSB; Cys-110 and Cys-187 that form the highly conserved disulfide bond; the ERY (134-136) conserved triplet that is very important for transducin activation in the visual phototransduction cascade; Lys-296, the site of 11-cis-retinal attachment; palmitoylated Cys-322 and Cys-323 that provide the constraint for the eighth helix in the cytoplasmic domain71.    2.7 The rhosg1 targets all homeologs generating more severe retinal degeneration phenotype. After determining that rhosg3 targets only one homeolog, while rhosg1 targets both homeologs, I realized there was an opportunity to analyze the specificity of Cas9, as well as to generate both heterozygous rhodopsin KOs and homozygous rhodopsin KOs. As the genome project for X. laevis is not complete, one of the initial caveats of this thesis work was that analysis of off-target effects could not be completed at the same depth as with other models. Comparing the editing efficiency of rhosg1 vs. rhosg3 allowed us to make some conclusions about potential off-target effects.  Single-cell in vitro fertilized embryos were injected with 6ng Cas9 mRNA, 700pg eGFP mRNA, and 3.7ng rhosgRNA, either rhosg1, rhosg3, or the non-targeting rhosgN. In vitro fertilized WT control embryos were injected with an equal volume of water (10nL). Tadpoles that arose from embryos injected with rhosg1 had, on average, 17-fold lower rod opsin values than the water-injected WT controls, while tadpoles that arose from embryos injected with rhosg3 had, on average, 9-fold lower rod opsin values than the water-injected WT controls (Fig. 2.G.a). Furthermore, there was a significant difference in rod opsin values between the tadpoles from embryos injected with rhosg1 versus those injected with rhosg3. There was no significant difference between tadpoles injected with rhosgN and the water-injected WT. In Fig. 2.G.b, there is significant RD in the contralateral eye collected from a tadpole that arose from an embryo injected with Cas9 mRNA and rhosg1. Most of the retina appears to be devoid of rod 	20		photoreceptors, while rods that are present have formed OSs that label with anti-rod opsin. Labeling with anti- rod transducin allowed us to detect rod photoreceptors that did not express rhodopsin. Transducin is dispersed throughout the photoreceptor when the retina is illuminated72, and as tadpoles are sacrificed with the retina illuminated, we hypothesized that transducin localization could serve as a method to identify rod photoreceptors that were rhodopsin KOs and would not label with anti-rod opsin. In Fig. 2.G.b, there is a photoreceptor in the rhosg1 retina that labels with anti- rod transducin, but very little anti-rod opsin is colocalized. Fig. 2.I.a shows a higher magnification image of this photoreceptor.  The standard indel analysis method in experiments with engineered nucleases is a Mismatch Cleavage Assay73. In CRISPR/Cas9 experiments, the region surrounding the sgRNA target-site is amplified by PCR from WT control genomic DNA (gDNA) and gDNA isolated from experimental tissue treated with Cas9 and sgRNA. These PCR products are denatured and hybridized for the formation of homoduplex (WT:WT or Mutant:Mutant) and heteroduplex (WT:Mutant) double-stranded DNA (dsDNA). Endonuclease digestion will cleave at the mismatch in heteroduplex dsDNA, and leave homoduplex dsDNA intact. The digest reactions are subsequently analyzed by gel electrophoresis. Initially, I attempted to analyze indels using this assay, however, the WT control samples were themselves cleaved, indicating the presence of natural mismatches generated from different rhodopsin alleles. Therefore, direct sequencing of PCR products amplified from gDNA of whole tissue was implemented for indel detection. Tadpoles with the most dramatically reduced rod opsin levels and most severe RD phenotypes were analyzed for indels by direct sequencing of 13-dpf tail gDNA (Fig. 2.G). Approximately 300bp of sequence around the rhosg1 and rhosg3 target-sites were amplified by PCR, analyzed by gel electrophoresis for band size, and sequenced by Sanger sequencing (Appendix A). We hypothesized that the presence of indels in the genome would result in truncated PCR amplicons, that when sequenced together with amplicons from unedited chromatin, would appear as background noise in the sequencing trace reads. In Figure 	21		2.G.d, there is an increase in background noise near the predicted cut-site in the PCR product from tail gDNA of a tadpole that arose from an embryo injected with Cas9 mRNA and rhosg1, while there is no evidence of background noise in gDNA from WT control tail tissue. Trace reads were sufficient for subjective observation of indels, however, when attempting to make comparisons between experimental animals, we found that visual inspection of trace reads was insufficient. Therefore, an excel algorithm was developed that compared the primary base call and secondary base call from *.abi files (generated by the ABI Prizm Sanger sequencing software and life technologies ab1 Peak Reporter online platform) for each position of a trace read to that of the WT trace read (Appendix J).  Using this algorithm, when the trace read calls the correct base for the primary peak, relative to the trace read of WT tissue from the same experiment, a value of 1 is assigned to that nucleotide position. When the trace read does not call the base correctly, but the secondary peak base is called the same as the WT, a value of 0.25 is assigned to that nucleotide position. Heterozygous positions in the WT sequence are assigned an alternate symbol in the trace read (Appendix J). If either the primary or secondary base called is the same as one allele, a value of 0.5 is assigned. If the primary and secondary bases represent both alleles, a value of 1 is assigned. If none of these criteria are met, a value of 0 is assigned. A Chi-squared test was utilized by comparing the direct sequencing trace of WT control gDNA versus the online reference sequences for each homeolog, to evaluate whether the injected tissue sequencing reads are significantly different from the WT control sequencing reads.  Plotted trace read graphs indicate that the sequence for homeolog Rho4L and both Rho4S alleles of JF847 and JF848 (rhosg1) are significantly different from the WT sequence, with the changes appearing near the predicted cut-site of rhosg1. The sequence for JF850 (rhosg1) is significantly different only at the Rho4S1-1 allele. The gDNA isolated from tail tissue of JF847 was further analyzed for detection of discrete indels, as the plotted graph was the most drastically different from WT. Interestingly, the moving average trend line for the Rho4S alleles 	22		of JF847 were very similar potentially indicating the same indel mutation that may have arisen from homologous recombination, either as a repair mechanism or not, early in development (Fig. 2.G.e).  Plotted trace read graphs for tadpoles that arose from embryos injected with rhosg3 and Cas9 mRNA indicated fewer instances of significant difference from the WT control sequence compared to the tadpoles that arose from embryos injected with rhosg1. Neither homeolog was significantly different from the WT in JF825, and only the Rho4L homeolog and Rho4S1-2 allele were significant in JF829 and JF830 (Fig. 2.G.f). The gDNA of JF829 and JF848 was further analyzed for presence of discrete indels based on the direct sequencing and histology results.  To analyze discrete indels, tail gDNA of JF829 (rhosg3), JF835 (WT), and JF848 (rhosg1) was amplified by the described homeolog-specific primers with homology-tails to the Bluescript-SKII+ (BS-SKII+) plasmid linearized with EcoRV. Clones were assembled into BS-SKII+ by Gibson Assembly74 and sequenced using the universal -21 primer. No clones sequenced from the WT sample had indels (Appendix K). 100% (5/5) of the clones from the Rho4L homeolog of JF848 had small deletions (Fig. 2.G.g) while only 40% (2/5) of the clones from the Rho4S1-1 allele had a deletion (Fig. 2.G.i). Notably, only two indel variants were amplified for the Rho4L homeolog, and only 1 indel variant was amplified for the Rho4S1-1 allele (120nt deletion). Potentially, this data indicates that editing occurred early in development and the animal may have a more uniform indel genotype. 20% (1/5) of the clones from the Rho4L homeolog of JF829 had deletions (Fig. 2.G.h) while no clones of the Rho4S homeolog showed presence of indels (Fig. 2.G.j). Most important, the edited allele for the Rho4L homeolog corresponded to the “matched” rhosg3, and the mismatched alleles were not edited. While sequencing the Rho4S1-1 allele of JF829, a mispriming event amplified an unedited Rho4S1-2 allele. This was the opportunity where I learned that there was a Rho4S1-2 allele in our animals and determined the primer sequence to amplify it separately from the Rho4S1-1. 	23		Only the Rho4S1-1 allele was analyzed for discrete indel clones as the primer for Rho4S1-2 was identified during the write-up of this thesis.  Whole embryo analysis by direct and discrete sequencing confirmed significant levels of editing within 24 hours of fertilization. gDNA of GFP+ 1-dpf whole embryo tissue was analyzed by direct and discrete sequencing (Fig. 2.H). Embryos injected with rhosg1 show significant sequence difference when compared to the WT sequence at both the Rho4L and Rho4S homeolog in 3/3 embryos sequenced (Fig. 2.H.a) while those that were injected with rhosg3 had significantly difference sequences at the Rho4L homeolog and Rho4S1-2 allele (matched allele) in 3/3 embryos sequenced, but the mismatched allele (Rho4S1-1) was not different (Fig. 2.H.b). Sequencing of discrete clones from rhosg1-1 showed that 100% (5/5) of Rho4L homeolog clones were edited (Fig. 2.H.c) and 80% (4/5) of the clones from the Rho4S1-1 allele were edited. All 5 of the indel mutations at the Rho4L homeolog were different, with two having insertions, only while two variants were amplified for the Rho4S1-1 allele both of which were deletions. Sequencing of discrete clones from rhosg3-2 showed that 100% (2/2) of Rho4L1-2 (matched allele) (Fig. 2.H.d) and 0% (0/3) clones from Rho4L1-1 (mismatched allele) had indels. None of the Rho4S1-1 (mismatched allele) discrete clones were edited in rhosg3-2. Expression data indicates that in X. laevis rhodopsin is not transcriptionally active until at least stage 3075,76,77, which corresponds to approximately 2-dpf, therefore, this evidence indicates that Cas9 may cleave heterochromatin.  	24		 Figure 2.G rhosg1 targets all homeologs generating more severe retinal degeneration phenotype. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by ANOVA showed a statistically significant difference between groups (P = 2.92 X 10-9).  Individual comparisons were subsequently made using Tukey test (P values shown on graph). The mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos injected with rhosg1 and rhosg3 were significantly different from the mean total rod opsin values of the WT. There was a statistically significant difference in the mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos injected with rhosg1 and rhosg3. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The tadpole retinas from embryos injected with rhosg3 were degenerated. The tadpole retinas from embryos injected with rhosg1 showed more severe retinal degeneration with patches of rods missing. (c) Sanger sequencing trace read of JF835 (WT). rhosg1 predicted cut-site highlighted in blue. (d) Sanger Sequencing trace read of JF847. rhosg1 predicted cut-site highlighted in blue. The presence of background peaks indicates mutations in the genome. (e) Graphed comparison of base call from direct 	25		sequencing of target-site for each nucleotide position of rhosg1-injected tail gDNA relative to WT control tail gDNA. Predicted cut-site is 3bp 5’ of PAM. Significance was evaluated by chi-squared test (P values shown on graph). JF847 and JF848 were different from the WT at both homeologs. JF850 was only different at the Rho4S1-1 allele. (f) Graphed comparison of base call from direct sequencing of target-site for each nucleotide position of rhosg3-injected tail gDNA relative to WT control tail gDNA. Predicted cut-site is 3bp 5’ of PAM. Significance was evaluated by chi-squared test (P values shown on graph). JF825 was not different from the WT at either homeolog. JF829 and JF830 were different from the WT at the Rho4L homeolog, and the matched Rho4S1-2 allele. (g) Sequencing results of discrete PCR amplicons from JF847 at the Rho4L homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 5/5 clones were edited. (h) Sequencing results of discrete PCR amplicons from JF829, at the Rho4L homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. Allelic variations have been merged in the WT reference with the usage of “K” at position 5 for “T” or “G”, “R” at position 34 for “A” or “G”, “M” at position 40 for “A” or ‘C”, and “R” at position 50 for “G” or “A”. 1/1 Rho4L1-2 (matched) clones were edited while 4/4 Rho4L1-1 (mismatched) clones were not. (i) Sequencing results of discrete PCR amplicons from JF847, at the Rho4S homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 2/5 clones were edited. (j) Sequencing results of discrete PCR amplicons from JF829, at the Rho4S homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. Allelic variations have been merged in the WT reference with the usage of “K” at position 5 for “T” or “G”, “W” at position 6 for “T” or “A”, “R” at position 34 for “A” or “G”, “M” at position 40 for “A” or ‘C”, and “R” at position 50 for “G” or “A”. 0/1 Rho4S1-2 (matched) clones were edited while 4/4 Rho4S1-1 (mismatched) clones were edited.     Figure 2.H rhosg1 targets all homeologs generating indels in injected embryos within 24 hours of fertilization. (a) Graphed comparison of base call from direct sequencing of target-site for each nucleotide position of rhosg1 injected embryo relative to WT control embryo. Sequencing Predicted cut-site is 3bp 5’ of PAM. Independence was evaluated by chi-squared test (P values shown on graph). 3/3 embryos sequenced were different from the WT at both homeologs. (b) Graphed comparison of base call from direct sequencing of target-site for each nucleotide position of rhosg3 injected embryo relative to WT control embryo. Predicted cut-site is 3bp 5’ of PAM. Independence was evaluated by chi-squared test (P values shown on graph). 	26		3/3 embryos sequenced were different at the Rho4L homeolog and Rho4S1-2 (matched) allele but not at the Rho4S1-1 (mismatched) allele. (c) Sequencing results of discrete PCR amplicons from rhosg1-1 of a, at the Rho4L homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 5/5 clones were edited. (d) Sequencing results of discrete PCR amplicons from rhosg3-2 of b, at the Rho4L homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. Allelic variations have been merged in the WT reference with the usage of “K” at position 5 for “T” or “G”, “R” at position 34 for “A” or “G”, “M” at position 40 for “A” or ‘C”, and “R” at position 50 for “G” or “A”. 2/2 Rho4L1-2 (matched) clones were edited while 3/3 Rho4L1-1 (mismatched) were not. (e) Sequencing results of discrete PCR amplicons from rhosg1-1 of a, at the Rho4S1-1 allele target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 4/5 clones were edited. (f) Sequencing results of discrete PCR amplicons from rhosg3-2 of b, at the Rho4S1-1 allele target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 5/5 Rho4S1-1 (mismatched) clones were not edited.    2.8 Germline transmission of edited rhodopsin gene. We examined progeny of a mating of two X. laevis derived from embryos injected with rhosg3 and Cas9 mRNA using techniques similar to those described above. We found reduced rod opsin in retinal extracts and retinal degeneration in contralateral eyes. Tail-derived gDNA had mutations corresponding to the predicted cut-site. Tadpoles injected with 6ng Cas9 mRNA and 2ng rhosg3 (section 2.5) were raised to sexual maturity, and siblings were mated for determination of germline transmission of Cas9-mediated indels. Female 2 and male 1 (F2M1) were mated on the same day that the WT embryos were generated for the experiment in section 2.9, therefore, these WT tadpoles, which arose from water-injected in vitro fertilized embryos, served as controls. Female 3 and male 1 (F3M1) were mated the next day. However, the F2M1 F1 tadpoles were noticeably smaller in size than the WT tadpoles, therefore, the rod opsin values may not be directly comparable. Although the F2M1 F1 tadpoles were smaller, they were otherwise normal in their morphology.  The total rod opsin values from the retinal extracts of 5 of 6 F2M1 F1 tadpoles, and the one F3M1 F1 tadpole, were less than 10% of average WT values while one F2M1 F1 tadpole had a value of 60% of WT (Fig. 2.I.a). Although this data suggested that these matings had generated 6 total homozygous rhodopsin KO tadpoles and 1 heterozygous rhodopsin KO 	27		tadpole, the retinas of the contralateral eyes showed many rod photoreceptors remaining that label with anti-rhodopsin. Therefore, it appears that the low rod-opsin value tadpoles likely have at least one functional rhodopsin allele. Nevertheless, the expression levels are low enough that the OS cannot form appropriately as they are short and bent (Fig. 2.I.b). Sequencing of the tail gDNA isolated from tadpoles JF929 (F2M1) and JF932 (F3M1) confirms the presence of mutations in the Rho4L homeolog. The trace read for JF932 appears almost identical to the trace read for JF929, suggesting that they contain the same indel mutation inherited from the male founder (Fig. 2.I.c). Direct sequencing of the Rho4S homeolog was not completed in time to include in this thesis thus neither the genotype of the F0 nor F1 animals was fully elucidated.  The tadpole JF926, which corresponds to the value of 60U on the dot blot, did not exhibit an obvious RD phenotype (Fig. 2.I.b). Potentially, this animal is WT with lower rhodopsin levels due to small size, or may be a less severe partial KO (e.g. 3 of 4 alleles present) with expression high enough that the outer segments still resemble WT. Direct sequencing of this animal, not performed in time to include in this thesis write-up, would confirm whether JF926 is WT or, in fact, a partial rhodopsin KO.   	28		Figure 2.I Germline transmission of edited rhodopsin gene. (a) Total rod opsin of retinal extracts from 15-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by t test showed a statistically significant difference between WT (from section 2.9) and rhosg3 KO Female 2 and Male 1 (P = 6.37 X 10-⁷). The total rod opsin values of retinal extracts of 5/6 tadpoles from Female 2 and Male 1, and the Female 3 and Male 1, were less than 10% relative to the mean total rod opsin value of the WT tadpoles. The total rod opsin value of retinal extracts from one Female 2 and Male 1 tadpole was 60% relative to the mean total rod opsin value of the WT. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. WT = JF917 (section 2.9), JF926 corresponds to the data point with a value of 60U in (a) of F2M1 group. JF929 corresponds to a data point near zero in (a) of F2M1 group. JF932 corresponds to the sole data point of F3M1 group in (a). The tadpole retinas label with anti-rhodopsin and anti-transducin indicating the presence of at least one functional rhodopsin gene. (c) Direct Sanger sequencing trace reads of Rho4L homeolog from JF929 (left) and JF932 (right) with rhosg3 predicted cut-site highlighted in blue. Similar background peaks indicate a shared indel inherited from Male 1.    2.9 The rhosg4 targets the last exon of rhodopsin inducing retinal degeneration.  It was necessary to design rhosg4, targeting the fifth and last exon of rhodopsin (Appendix A), in order to test whether heterologous expression of rhodopsin-GFP is possible (section 3.1). However, we realized that rhosg4 might also be utilized to establish endogenous X. laevis dominant-RP models, analogous to mutations such as human Q344ter, a premature stop codon that terminates at the glutamine residue of the QVS(A)PA C-terminal localization signal required for exclusive localization of rhodopsin to the outer segment25,78.  Single-cell in vitro fertilized embryos were injected with 4.5ng of Cas9 mRNA, 700pg of eGFP mRNA, and 1.5ng of rhosg4; GFP+ embryos were raised until 15-dpf. We did not necessarily expect a decrease in rhodopsin expression in these animals as there would be no reduction in mRNA levels as a result of nonsense-mediated decay79. Rather lower rod opsin values would likely indicate death of rod photoreceptors as a result of mislocalization, misfolding, or other toxicity. The average rod opsin value of retinal extracts isolated from tadpoles that arose from embryos injected with rhosg4 were significantly different from the WT (Fig. 2.J.a).  Tadpole JF960 exhibited severe RD in the central retina, with less RD in the periphery. Because the retina increases in size by addition of peripheral cells, the peripheral photoreceptors are younger and in this animal, have not yet died (Fig. 2.J.b). This data indicates 	29		that deletions at this target-site may potentially produce endogenous X. laevis dominant-RP models, although in this particular case we did not observe significant mislocalization of rhodopsin to inner segments, and demonstration of a dominant phenotype would likely require breeding. Sequencing data was not gathered in time to include in this thesis.   Figure 2.J rhosg4 targets the last exon of rhodopsin inducing retinal degeneration.  (a) Total rod opsin of retinal extracts from 15-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by Mann-Whitney U test showed a statistically significant difference between WT (from section 2.9) and rhosg3 KO Female 2 and Male 1 (P = 0.030).  (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The tadpole retinas from embryos injected with rhosg4 were more severely degenerated in the central retina than in the peripheral.   2.10 Outer segments of rhodopsin knock-out rod photoreceptors are malformed. Researchers have reported that in rhodopsin KO rod photoreceptors there is an attempt to form an OS, but the structure is severely malformed80. In Figure 2.K.a shows an example of a rod photoreceptor in the retina of a tadpole from an embryo injected with rhosg1 and Cas9 mRNA that does not label with anti-rhodopsin, with the exception of two small spots in the OS structure indicated by white arrows. Rhodopsin-less cells were rare, suggesting that complete loss of rhodopsin expression kills rods rapidly. Anti-rod transducin labeling confirms that this is a rod photoreceptor. Fig. 2.K.a is a higher magnification image of the retina in 2.G.b.   	30		2.11 Mislocalized rhodopsin proteins likely indicate in-frame mutations.  CRISPR/Cas9-mediated indel mutations may produce in-frame deletions that retain or gain functionality and produce non-KO phenotypes81. In Fig. 2.K.b, anti-rhodopsin labels mislocalized small punctate structures (representative examples indicated by white arrows) throughout the rod photoreceptor, likely indicating an in-frame mutation that interferes with rhodopsin biosynthesis.   Figure 2.K Observations made on morphology. (a) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay in section 2.7 (JF848). White arrows indicate two small points in the outer segment that still label with anti-rhodopsin.  (b) Representative cryosectioned and immunolabeled contralateral eye from tadpoles used in dot blot assay in section 2.2 (JF27). White arrows indicate punctate/mislocalized structures that label with anti-rhodopsin.   	31		3. CRISPR/CAS9-MEDIATED INSERTION OF EXOGENOUS DNA 3.1 Heterologous expression of rhodopsin-GFP fusion protein through injection of rhosg4 with repair template containing eGFP sequence and rhodopsin homology.   To test whether HDR approaches for heterologous expression in X. laevis are possible, a repair template was constructed with approximately 1200 bp of homology around the rhosg4 predicted cut-site, in the last exon of rhodopsin (Fig. 2.F.a), encoding eGFP at a proven position in the rhodopsin peptide sequence that would express a functional rhodopsin-GFP fusion protein after integration by HDR82. The homology sequence on the repair template also encodes a mutagenized rhosg4 target-site to prevent re-cleavage by Cas9 after integration by HDR. A more detailed description of construct design can be found in Appendix E.   Single-cell in vitro fertilized embryos were injected with 6 ng Cas9 mRNA and 3.7 ng rhosg4. Varying amounts of repair template, both linear and plasmid, were tested: 1ng plasmid DNA, 1ng linear (linearized by SmaI), 500pg of both plasmid and linear (250pg of each), and 100ng of both plasmid and linear (50pg of each). 1ng, of either plasmid or linear, and 500pg of template proved very toxic while 100pg of repair template was not toxic, and survival rates were comparable to uninjected in vitro fertilized WT controls (Fig. 3.A). Needle plugging issues during injection of the 1ng linear template likely resulted in a lower dose of both RNA and repair template, thus, survival rates were comparable to 100ng/embryo of repair template. Eyes of injected tadpoles were screened by fluorescence microscopy during development, and two tadpoles were identified with GFP+ eyes (Fig. 3.A.b), tadpole JF851 was found in the group that was injected with 100pg of repair template while JF852 arose from an embryo injected with 1ng circular repair template. Both eyes of each tadpole were GFP+, therefore, each eye was collected for cryosectioning and immunolabeling at 10-dpf. Examined eyes exhibited significant RD, and GFP+ photoreceptors were more common at the periphery of the retina (i.e. developmentally younger cells), potentially due toxicity of rhodopsin-GFP protein alone or in 	32		combination with KO of other homeologs/alleles. GFP was localized to rod OSs, consistent with literature reports for similar rhodopsin-GFP fusion proteins 82 83.   Interestingly, while screening GFP+ eyes during development, some tadpoles exhibited GFP+ cells elsewhere in the body (Fig. 3.A.d), suggesting the tendency for the repair template to integrate randomly. Tadpoles that had GFP+ retina did not present with this phenotype, and only a few of those tadpoles that survived the 1ng dosage of circular repair template exhibited GFP+ cells elsewhere in the body. Attempts to amplify the integrated eGFP sequence by PCR were unsuccessful.  Figure 3.A Heterologous expression of rhodopsin-GFP fusion protein through injection of rhosg4 with repair template containing eGFP sequence and rhodopsin homology. (a) Cryosectioned and immunolabeled retina with GFP expression colocalized to outer segments of rod photoreceptors with rhodopsin. The degenerated tadpole retinas show GFP signal colocalized with rhodopsin in outer segments. (b) Above: JF851, 5-dpf with 	33		fluorescent retina visualized externally. Below: Tadpole expressing GFP in abdominal cells. (c) Survival rate of embryos injected with varying amounts of repair template. Only those injected with 100pg template showed a similar rate of survival comparable to uninjected embryos.    3.2 Heterologous expression of rhodopsin with novel M13F epitope through injection of rhosg1 with repair template containing rhodopsin homology with a missense mutation.  I designed a second repair template construct to test whether Cas9 and repair template delivery can facilitate HDR and generation of a missense mutation in X. laevis. A more detailed description of construct design can be found in Appendix F. This construct contained approximately 1500bp of homology centered on the rhosg1 predicted cut-site and was designed to introduce a novel missense mutation, M13F, detected by antibody 2B2, via alteration of 2 bp in the rhodopsin gene. In addition, the homology in the repair template contained a mutant rhosg1 with 2bp silent mutation for prevention of subsequent targeting and cleavage.  Single-cell in vitro fertilized embryos were co-injected with 700pg eGFP mRNA to visually confirm RNA injection, sacrificed at 14-dpf, with one eye collected for dot blot immunoassay and the other for cryosection and immunolabeling. The dot blot using A5-3 antibody for M13F epitope detection proved to be uninformative, although control M13F samples (M13F rhodopsin expressed in COS cells) gave intense signals, suggesting that the efficiency was low (data not shown). Six eyes were cryosectioned, one of which contained a small number of 2B2-positive rods (Fig. 3.B.a), while the other eyes did not. Many of the eyes examined exhibited severe RD, and therefore potentially the editing from rhosg1 is too toxic to the retina, and the infrequent repair by recombination events does not save the retina from degeneration. The eye containing 2B2-positive rods, from the tadpole “JF866”, did not exhibit any RD, therefore, potentially there were fewer editing events and the cells survived for positive identification. 	34		 Direct sequencing of the Rho4L homeolog in JF866 was employed in order to confirm the integrated M13F mutation. Primers were designed to amplify outside the homology arms to prevent amplification of residual template DNA. PCR products of tail gDNA from an uninjected in vitro fertilized WT and from JF866 were sequenced, and trace reads did not indicate the presence of a second peak relative to the desired M13F mutation in JF866 (Fig. 3.B.c), nor did they indicate the presence of background noise at the predicted cut-site (Fig. 3.B.d).   Figure 3.B Heterologous expression of rhodopsin with novel M13F epitope through injection of rhosg1 with repair template containing rhodopsin homology with missense mutation. (a) Cryosectioned and immunolabeled retina with 2B2 anti-rhodopsin, specific to the M13F epitope. Two rods have outer segments that label with 2B2 indicating heterologous expression of an M13F in rhodopsin. (b) Survival rates of GFP+ (dotted-line) and GFP- (solid line) injected embryos from fertilization to 14-dpf. GFP- embryos were destroyed 3-dpf. Only those injected with 100pg of template showed similar levels of survival compared to WT.  (c) Direct sequencing trace reads of WT (above) and JF866 (below). Highlighted peaks indicate where the M13F mutation was designed, but is not present. (d) Direct sequencing trace reads of WT (above) and JF866 (below). Highlighted peaks indicate where the silent mutation in rhosg1 was designed, but the lack of background peaks indicates a lack of editing.           	35		4. RNA-GUIDED CAS9 GENE DRIVE SYSTEM 4.1 Confirming enzymatic activity of Cas9-GFP and Cas9-FLAG by mRNA injection. Construction of a transgene Cas9 expression cassette, with Cas9 and sgRNA encoded, would enable tissue-specific or inducible expression allowing a wider range of gene KO possibilities and investigative questions. We compared three Cas9 variants (Cas9, Cas9-FLAG, Cas9-GFP) by mRNA injection for confirmation that Cas9 fusion proteins had catalytic activity. Cas9-FLAG mRNA was in vitro transcribed from pMLM3639, and Cas9-GFP was PCR amplified, with a T7 promoter encoded on the forward primer, and PCR amplification product served as in vitro transcription template. Single-cell in vitro fertilized embryos were injected with 6ng of one of the variant Cas9 mRNAs and 3.7ng of either rhosg3 or rhosg1. All embryos were injected with 700pg of eGFP mRNA and screened for GFP-fluorescence 1-dpf. Retinal extracts of tadpoles that arose from embryos injected with Cas9 mRNA and Cas9-FLAG mRNA, either injected with rhosg1 or rhosg3, showed significantly lower rod opsin values than tadpoles that arose from uninjected WT control embryos. Rod opsin value in retinal extracts of tadpoles from embryos injected with Cas9-GFP mRNA were not different from the uninjected WT controls.  However, further investigation of histology phenotypes indicated some RD in the analyzed eyes of tadpoles that arose from embryos injected with Cas9-GFP mRNA and rhosg1. Sequencing of 1-dpf whole embryos injected with Cas9-GFP mRNA and rhosg1 indicated that while editing frequency was low, there were indels present in two of five discrete clones sequenced. Cas9-GFP was selected to be encoded on the Cas9 cassette, as GFP-positive confirmation would prove the simplest positive control for transgene expression, and we anticipated that even low Cas9-GFP activity would eventually result in complete editing given that expression was not expected to be transient.  	36		 Figure 4.A Confirming enzymatic activity of Cas9-GFP and Cas9-FLAG by mRNA injection. (a) Total rod opsin of retinal extracts from 14-dpf tadpoles quantified by anti-rod opsin dot blot assay. Data analysis by ANOVA showed a statistically significant difference between groups (P = 8.65 X 10-10). Individual comparisons were subsequently made using Tukey test (P values shown on graph). The mean total rod opsin values from retinal extracts of whole tadpole eyes from embryos injected with Cas9 mRNA with rhosg1 or rhosg3 and Cas9-FLAG mRNA with rhosg1 or rhosg3 were significantly different from the mean total rod opsin values of the WT. There was not a statistically significant difference between the mean total rod opsin values of those injected with Cas9-GFP versus the WT. (b) Representative cryosectioned and immunolabeled contralateral eyes from tadpoles used in dot blot assay. The tadpole retinas from embryos injected with rhosg1 showed a more severe retinal degeneration than the retina of a tadpole from an embryo injected with Cas9-GFP. Retinal degeneration of the tadpole from an embryo injected with Cas9-GFP mRNA indicates catalytic activity. (c) Sequencing results of discrete PCR amplicons from whole embryo 24hrs after injection with Cas9-GFP mRNA and rhosg1, at the Rho4L homeolog target-site region. PAM is in red, target-site is bolded in WT reference sequence in the first row. 2/5 clones were edited indicating catalytic activity of Cas9-GFP.    	37		4.2 A rod opsin-promoter driven Cas9-GFP, and an X. tropicalis U6 promoter-driven sgRNA. Transgenic expression of a Cas9 cassette would facilitate tissue-specific editing, for example by using a rod opsin promoter (XOP) to drive expression in rod photoreceptors, and also inducible editing, for example by using a heat-shock promoter (HSP70). Tissue-specific or inducible editing would allow generation of KOs that would otherwise be lethal.   A Cas9 cassette encoding XOP expression of Cas9-GFP and X. tropicalis U6 (xtU6) expression of sgRNA was cloned into the standard transgene construct used in our lab84. A more detailed description of construct design can be found in Appendix H. XOP has very high transcription rates, and is transcriptionally active in rod photoreceptors only, therefore we generated XOP-Cas9-GFP-xtU6 transgenics to assay for Cas9-GFP toxicity at XOP expression levels. Death of rod photoreceptors would indicate XOP-Cas9-GFP toxicity. These transgenics did not encode a target-site on the rhosgRNA backbone sequence (sgBB) encoded on the transgene. Transgenesis injection protocol was performed as described by Tam et al.85. We also generated XOP-Cas9-GFP-xtU6 transgenics encoding rhosg3. Retinas of transgenics, both sgBB and rhosg3, were mosaic with sparse GFP+ rod photoreceptors. Malformation of GFP+ rod OS were not observed in the rhosg3 transgenics, indicating that editing of rhodopsin homeologs had not occurred (Fig. 4.B). Furthermore, although the Cas9-GFP does contain N-terminal and C-terminal nuclear-localization-signals (NLS), Cas9-GFP appeared to be dispersed throughout the inner and outer segments of the rod photoreceptors, with higher intensity in inner segments. The dispersion of the Cas9-GFP throughout the photoreceptor could explain the lack of editing as the Cas9 should be localized to the nucleus for maximum DNA binding and editing activity.  A potential confounding factor is that our standard transgene constructs are linearized with FseI.  However, as there is a second FseI site in the Cas9-GFP in this experiment, the construct was linearized with AflIII, removing one of the 3’ insulator sequences. This could 	38		account for the mosaic phenotype observed in all animals. The second FseI site in Cas9-GFP was mutagenized (Appendix L) as a silent mutation for future experiments.   Figure 4.B A rod opsin-promoter (XOP) driven Cas9-GFP, and an X. tropicalis U6 promoter-driven sgRNA. (a) Cryosectioned and immunolabeled retinas of transgenic 14-dpf tadpole expressing non-targeting sgRNA backbone (above) and rhosg3 (below). The rod OS are not malformed therefore editing likely did not occur. (b) 80X magnification of cryosectioned and immunolabeled retinas of transgenic 14-dpf tadpole expressing non-targeting sgRNA backbone (left) and rhosg3 (right). GFP signal is dispersed throughout the cell body and not localized to the nucleus.    4.3 A CMV-promoter driven Cas9-GFP, and human U6 promoter-driven sgRNA.  A second tested transgene construct incorporated a cytomegalovirus (CMV) promoter-driven Cas9-GFP and a human U6 (hU6) promoter driven sgRNA. The hU6 promoter has been demonstrated to be functionally equivalent to the Xenopus U6 promoter in X. laevis oocytes86. A more detailed description of construct design can be found in Appendix I. This construct was tested with both rhosg1 and rhosg3, compared to an empty sgRNA backbone sequence (sgBB). Embryos that were uniformly GFP-positive 24hours after injection were raised and analyzed. Eyes analyzed indicated low uniform expression throughout the eye, with sparse cells exhibiting higher intensity GFP expression (Fig. 4.C.a). GFP localization was again evident throughout the outer and inner segment of rod photoreceptors, with higher intensity in the inner segment (Fig. 4.C.b.). One cell, with very high-intensity GFP, when viewed at 80X magnification indicated localization to what appears to be the nucleolus (Fig. 4.C.b). Photoreceptors that were GFP+ 	39		had normal OS morphology, indicating a lack of Cas9 editing. As previous experiments confirmed the activity of Cas9-GFP mRNA when injected (Fig. 4.A.c), the absence of RD in GFP+ photoreceptors indicated an issue with the sgRNA component of the CRISPR editing technique. Reverse-transcriptase PCR (RT-PCR) indicated that rhosg1 was not expressed at detectable levels (Fig. 4.C.c). Details of RT-PCR can be found in section 5.6. Furthermore, direct sequencing of gDNA from rhosg1 transgenic embryos did not indicate presence of indels (Fig. 4.C.d). Future investigations will employ an alternate transgenic construct87.  Figure 4.C A CMV-promoter driven Cas9-GFP, and human U6 promoter-driven sgRNA.  (a) Cryosectioned and immunolabeled retinas of transgenic 14-dpf tadpole expressing non-targeting sgRNA backbone (top), rhosg1 (middle), and rhosg3 (bottom). The rod OS are not malformed therefore editing likely did not occur. (b) Photoreceptor in retina from transgenic expressing non-targeting rhosgRNA with a higher intensity GFP signal localized inside the nucleus. (c) RT-PCR products from total RNA of WT eye tissue or eye tissue of CMV-Cas9-GFP-hU6rhosg1 transgenic tadpoles (TG). cDNA templates were prepared from total miRNA and mRNA by incubation of RNA isolates with reverse-transcriptase (+RT) or without (-RT). pDNA of the CMV-Cas9-GFP-hU6rhosg1 transgene construct (P) served as a positive control for “rhosg1” primers. The “RHO” primer pairs were designed to span exon1 and exon2 of RHO coding sequence. A band corresponding to the expression of rhosg1 (rhosg1 TG +RT) was not present. (d) Direct sequencing of gDNA collected from 1-dpf CMV-Cas9-GFP-hU6sgBB (top row) and CMV-Cas9-GFP-hU6rhosg1 (bottom three rows) embryos at the rhosg1 target-site on homeolog Rho4L. All sequenced were identical to the WT therefore editing likely did not occur.      	40		5. METHODS 5.1 Cas9 and eGFP mRNA In Vitro Transcription Cas9 mRNA was in vitro transcribed using pMLM3613 (Addgene plasmid #42251), linearized by PmeI, as template DNA with T7 mMessage mMachine Ultra kit, from Ambion, or HiScribe ARCA kit from NEB. These kits include the Anti-Reverse Cap Analog (ARCA) that allows for synthesis of  RNAs capped exclusively in the correct orientation. Approximately 5µg of plasmid DNA (pDNA) was linearized with PmeI and then ethanol precipitated for removal of restriction digest buffer salts and concentration of template DNA. Complete linearization of pDNA was confirmed by agarose gel electrophoresis. The digest reaction was transferred to a nuclease-free 1.5mL eppendorf tube for ethanol precipitation. The DNA pellet was resuspended in 8.7µL of nuclease-free H2O then quantified and evaluated for purity by nanodrop. Approximately 1.5-2µg of template DNA was added to the in vitro transcription reaction, and incubated for 1 hour. The reaction was incubated for 15 minutes with DNaseI for degradation of template DNA, followed by a 40minute incubation with a poly-a tailing enzyme for addition of polyA tails to mRNA transcripts. The reaction was cleaned-up using by column purification with Qiagen RNeasy kit. If two reactions were completed in parallel, they were combined by ethanol precipitation with nuclease-free ammonium acetate as the salt and resuspended in 21µL nuclease-free H2O. Final products were evaluated for size and quality by agarose gel electrophoresis, and absorbance at 260nm was measured using the Thermo Scientific NanoDrop 2000c Spectrophotometer for quantification of RNA. RNA was stored at -80°C immediately after purification. pMLM3639 (Addgene plasmid #42252) was used for in vitro transcription of Cas9-FLAG mRNA. pMLM3613 and pMLM3639 were gifts from Keith Joung.    	41		5.2 Cloning sgRNA target-site sequences into sgRNA backbone of pDR274  Approximately 5µg of pDR274 was linearized with BbsI, dephosphorylated with Calf Intestinal Alkaline Phosphatase and gel extracted for purification and evaluation of digest completion. 1µL of each oligo, synthesized with 5’ phosphorylation, were combined in a final volume of 10µL ROH2O, held at 98°C for seven minutes, allowed to cool to RT for thirty minutes, and held on ice for 10 minutes to hybridized ssDNA into dsDNA. 100ng of linearized pDR274, vector, and 540pg (1.82µL of a 1/500 dilution) of hybridized oligos, insert, were ligated with a T4 ligase reaction at RT. 1 µL of ligation reaction was added to 4µL of TE buffer, combined with 45µL chemically competent alpha-DH E. coli cells, incubated on ice 30 minutes, held at 42°C for 30 seconds, transferred to ice for 10 minutes, after which 500uL LB was added and cells were shaken at 225 RPM, 37°C for 1hr. Cells were then spun down at 8K for 2 minutes, 400µL supernatant removed, and remaining plated on LB+AMP.   5.3 In Vitro Transcription of sgRNA  sgRNA was in vitro transcribed using pDR274 (Addgene plasmid # 42250) (with desired sgRNA cloned in and linearized by DraI) as template DNA with the MAXIscript kit from Ambion. Approximately 5µg of plasmid DNA (pDNA) was linearized with DraI and then ethanol precipitated for removal of restriction digest buffer salts and concentration of template DNA. Complete linearization of pDNA was confirmed by agarose gel electrophoresis. The digest reaction was transferred to a nuclease-free 1.5mL eppendorf tube for ethanol precipitation. The DNA pellet was resuspended in 12.7µL of nuclease-free H2O then quantified and evaluated for purity by nanodrop. Approximately 1.5-2µg of template DNA was added to the in vitro transcription reaction, and incubated for 1.5 hours. The reaction was incubated for 15 minutes with DNaseI for degradation of template DNA. The reaction was cleaned-up using by column purification with the Qiagen miRNeasy kit. If two reactions were completed in parallel, they were 	42		combined by ethanol precipitation with nuclease-free ammonium acetate as the salt and resuspended in 21µL nuclease-free H2O. Final products were evaluated for size and quality by agarose gel electrophoresis, and quantified by nanodrop. RNA was stored at -80°C immediately after purification. pDR274 was a gift from Keith Joung.   5.4 RNA Microinjection   Two WT females X. laevis were injected with 100 units of human chorionic gonadotropin (HCG) two days prior to the microinjection to lay eggs. The day before the microinjection experiment, both females were injected with 70 units HCG. The day of the experiment, a WT fertile male was chosen based on darkness of mating pads, anesthetized in 0.1% Tricane, 0.1XMMR until unresponsive to physical stimulation. His heart was dissected out according to ethical requirements for double euthanization, and his testes were removed and stored in 1X MMR on ice. Eggs were manually expelled from females into empty petri dish. Once enough eggs collected, in vitro fertilization proceeded. One-half to 1/3 of one teste was mashed in 250µL 0.1XMMR for release and activation of sperm. Sperm were dribbled over eggs in petri dish, allowed to incubate for 2 minutes, followed by submergence of eggs in 0.1XMMR and incubation for 20 minutes. Embryos were visually inspected for signs of activation and therefore fertilization. After 20 minutes in 0.1XMMR, 2% cysteine (pH 8, 1X MMR) was pipetted over embryos for removal of follicle cell sheath. Defolliculated embryos were arranged in monolayer in 2% agarose injection plates, buffered with 0.4X MMR, 6% Ficoll. RNA reactions were spun at RT, 13,000 RPM prior to loading into a pulled glass micropipette with 20-25µm diameter, and back primed with mineral oil. A Hamilton syringe pump was set to 36µL per hour, with continuous flow. Embryos were injected for 1 second equating to 10nL of RNA reaction. After microinjection, embryos were transferred to 18°C for approximately 2.5 hours until the four cell 	43		stage, at which point the embryos that exhibited appropriate signs of cell division were transferred to 0.1XMMR + 6% Ficoll and stored overnight at 18°C.   The day after injection, the healthy embryos are transferred to 0.1XMMR, while the dead are removed. A small number of GFP+ dysmorphic embryos were prepped for extraction of gDNA for sequencing analysis of indels. Embryos were screened for GFP+ phenotype for three days, upon which GFP- embryos were euthanized by ethyl 3-aminobenzoate methanesulfonate salt (tricaine) overdose and the GFP+ embryos were transferred to 0.1XMMR.   On the 14th day of development, stage 48, tadpoles were sacrificed by pithing, one eye was dissected out and solubilized according to standard lab protocols88 for quantification of rod opsin protein by immunoassay dot blot. The tail was removed for genomic prep and extraction of gDNA for sequence analysis. The the other eye was fixed in 4% paraformaldehyde overnight. The fixed eye was then embedded in OCT, cryosectioned and immunolabeled.  Indel analysis consisted of direct sequencing of whole tissue for to asses for perturbation of sequencing trace near the predicted cut site, and discrete sequencing of individual clones for confirmation of random insertions or deletions (indels). For direct sequencing, rhodopsin target-sites were amplified using primer sequences in Appendix A. PCR products were cleaned-up by bead purification or exonuclease-SAP digestion and sequenced with the respective forward primers by Sanger dye-termination method. Rho4L PCR conditions: 98°C for 5 minutes, 35 cycles of 98°C for 10s, 63°C 10s, 72°C 10s, with a final two-minute extension at 72°C, and held at 4C. Rho4S conditions were the same with annealing temperature at 58C. Sequence of discrete clones was performed using the same loci-specific primers with homology tails (F:GTCGACGGTATCGATAAGCTTGAT, R:TCCCCCGGGCTGCAGGAATTCGAT), and cloning the resulting PCR products into Bluescript-SKII+ linearized at EcoRV via Gibson Assembly. PCR conditions: 98°C for 5 minutes, 3 cycles of 98°C for 10s, 63°C 20s, 72°C 10s, 32 cycles of 98°C 10s, 69°C (Rho4L) or 58°C (Rho4S), 72°C 10s, with a final two-minute extension at 72°C, 	44		and held at 4°C. Individual PCR amplicons were cloned into linearized BS-SKII+ by Gibson Assembly 74, and sequenced using -21M13 forward universal primer. Gibson assembly reaction conditions: ~10ng linear vector (0.22uL), ~10ng PCR product (0.98uL), 4uL Gibson Assembly Master Mix, incubate 50°C for 60minutes, followed by 4°C hold. Transform into chemically competent cells, miniprep and send for sequencing.   5.5 HDR Specific Methods  HDR repair fragments were cloned into the Bluescript-SKII+ plasmid backbone. Construct details are found in Appendix E (eGFP-HDR), and Appendix F (M13F-HDR). Rhodopsin homology was amplified from gDNA using primers based on the rhodopsin gene sequence published by Batni et al.69 (identical to the Rho4L1-1 allele). The sgRNA target-sites were subsequently mutagenized, as silent mutations, within 5 nucleotides of the PAM by quickchange mutagenesis.   Repair template was purified by Qiagen miniprep without RNase added to P1 buffer. A prep was linearized with SmaI, and both the circular preps and linear preps were concentrated using QIAEX II bead purification kits. Final stocks of repair template were incubated 20 minutes on ice with RNA ladder, and then analyzed by agarose gel electrophoresis to confirm no RNase contamination. RNA ladder was not degraded and bands were easily identified the same as the control incubation with DEPC-treated water.  Embryos were injected with 6.6ng Cas9 mRNA, 4ng/embryo rhosg1 or rhosg4. Embryos injected with the eGFP-HDR construct were not co-injected with eGFP mRNA. Embryos injected with M13F-hDR construct were co-injected with 700pg eGFP mRNA. WT controls were injected with sterile water.  Variable amounts of repair template were injected for comparison: 500pg/embryo (250pg linear, 250pg circular uncut plasmid), 100pg/embryo (50pg linear, 50pg circular), 1ng/embryo 	45		circular, and 1ng/embryo linear. 500pg/embryo and greater proved to be quite toxic to the embryos, while survival rate of embryos injected with 100pg were not different from WT controls. eGFP-HDR animals were screened using an epifluorescence-equipped dissecting microscope for GFP-positive eyes. M13F-HDR animals were screened for GFP-positive phenotype as described in RNA-injection protocol and sacrificed at 14 days. One eye was solubilized according to standard lab protocols88 while the other eye was collected for cryosection and immunolabeling. Dot blot was performed using the A5-3 monoclonal antibody, sensitive to M13F epitope by dot blot, and B630N for evaluation of RD as in RNA-injection experiments. In the M13F-HDR experimental animals, the contralateral eye was sectioned and labelled with 2B2 (specifically labels rhodopsin containing an M13F mutation). Both eyes of the eGFP-HDR animals were cryosectioned and immunolabeled for confirmation of rhodopsin-GFP localization and expression.   5.6 Transgenesis Specific Methods Transgenesis methods were carried out as described in Tam et al.85. RT-PCR was performed using Qiagen miRNeasy for isolation of total RNA transcripts from solubilized whole GFP+ tadpoles, and miScript RT II kit for cDNA synthesis from both miRNA and mRNA using the provided HiFlex buffer. 1µg of RNA was used as template and incubated with provided nucleic mix and RT mix for 60minutes at 37°C, then 5 minutes at 95°C. “RHO” primers, that span exon 1 and exon 2, were designed to amplify from rhodopsin as a positive control for cDNA synthesis. 1µg of RNA was also incubated with nucleic mix and buffer without RT to control for false-positive amplification from genomic DNA. “rhosg1” primers amplify the cDNA prepared from rhosg1 miRNA. “RHO” primer products have an expected band size of 248bp, while “rhosg1” primer products have an expected band size of 104bp. The transgene construct was utilized as a positive control for “rhosg1” primers. PCR conditions: 95°C 1min, 35 cycles of 	46		95°C for 20s, 64°C (RHO) or 60°C (rhosg1) for 15s, and 68°C for 30s, followed by 5min at 68°C, and 4°C hold.                                                	47		CONCLUSION Generation of first generation KO phenotypes in X. laevis is possible by RNA injection of single-celled in vitro fertilized embryos, increasing the utility of X. laevis as a model organism, as previously, a reliable method for KO and KD was not available. The optimal conditions for injection included 6ng of Cas9 mRNA, 700pg of eGFP mRNA, and 3.7ng of rhodopsin-targeting sgRNA per embryo and raising at 18°C. Enrichment by screening for GFP+ embryos at 1 to 3-dpf resulted in more severe RD phenotypes, including lower rod opsin retinal extract values and malformed/missing rod OS.  As the X. laevis genome project is not complete, comparing the indel frequency and RD phenotypes between tadpoles of embryos injected with rhosg1 vs. rhosg3 provided a method for determination of Cas9 specificity. My data indicates that Cas9 is highly specific as indels were only identified in the discrete clones amplified from the alleles with perfectly matched target-sites to rhosg3, Rho4L1-2 and Rho4S1-2, and no indels were ever identified from discrete clones of the Rho4L1-1 and Rho4S1-1 alleles with two mismatches to rhosg3 at positions 14 and 20 distal to the PAM. Injection of the control non-targeting rhosgN resulted in healthy retinal morphology and rod opsin values that were not significantly different from WT. rhosg1 generated the most severe RD phenotypes with significantly lower values of rod opsin content from retinal extracts compared to both WT and those injected with rhosg3. Sequencing analysis of indels in 1-dpf embryos indicated that editing occurs early in development, as well as showing evidence that editing of transcriptionally inactive rhodopsin is possible. Rod opsin content assayed by dot blot and phenotype of the contralateral eye from the same tadpole did not always positively correlate, indicating that editing likely results in chimeric tadpoles with a wide variety of indels in one animal. Therefore, as germline transmission of Cas9-mediated indels at the rhosg3 target-site through inbred matings was successful, generation of pure F1 lines for further investigations is ongoing. Heterozygous and homozygous rhodopsin KO X. laevis lines 	48		would be the first of their kind, and there are several interesting investigations, such as relative expression levels of homeologs, that could be initiated.  Co-injection of RNA with homology repair templates successfully generated tadpoles that expressed a rhodopsin-GFP fusion protein in rod photoreceptors localized to the OS with endogenous rhodopsin, and tadpoles that expressed the novel M13F epitope in the endogenous rhodopsin protein, localized to the OS. Success frequency was low, however, it is interesting that both eyes were GFP+ in those tadpoles that were positive for heterologous expression of the rhodopsin-GFP. This evidence likely indicates that in each of these tadpoles two independent HDR events occurred during development, as editing in the single-celled phase of embryogenesis does not appear to occur in our experiments with Cas9 mRNA, and the animals also possessed non-GFP expressing rods in both eyes. The low likelihood of this occurring randomly suggests that conditions for HDR were in some way substantially more favorable in these particular animals, and that the success rate for HDR could be substantially increased by optimization. This technique provides an interesting opportunity for generation of point mutations in the rhodopsin gene linked to the M13F epitope as a reporter. Introduction of point mutations provides a very exciting opportunity for generation of multiple different genetic models of RP or investigation of rhodopsin properties. Current transgenesis techniques allow modeling by expression of dominant mutant rhodopsins. Using this HDR approach, it would be possible to generate pure genetic models of multiple forms of RP in X. laevis for the first time.  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PMCID: PMC4180472  	56		APPENDICES APPENDIX A Sequences for each allele target-site with respective primer sequences for amplification.  rhosg1, rhosg2, rhosg3 are located within the first exon of rhodopsin. The start codon of rhodopsin includes the 5’ “T” and “G” of rhosg3. Primers are also located within exon 1. The PAM adjacent to rhosg2 is abolished in Rho4L1-1 and Rho4S1-1. rhosg4 is located in exon 5, primers for amplification had not been optimized upon write-up of this thesis.   START CODON RHOSG3 RHOSG3vA RHOSG2 RHOSG1 RHOSG4 STOP CODON  Rho4L1-1 Target-site (rhoPCR1F:CAGTTGGGATCACAGGCTTC, rhoPCR1R:CAGGATGTAGTTTAGGGGTG) TAGGGATCCTTTGGGCAAAAAAGAAACACAGAAGGCATTCTTTCTATACAAGAAAGGACTTTATAGAGCTGCTACCATGAACGGAACAGAAGGTCCAAATTTTTATGTCCCCATGTCCAACAAAACTGGGGTGGTACGAAGCCCATTCGATTACCCTCAGTATTACTTAGCAGAGCCATGGCAATATTCAGCACTGGCTGCTTACATGTTCCTGCTCATCCTGCTTGGGTTACCAATCAACTTCATGACCTTGTTTGTTACCATCCAGCACAAGAAACTCAGAA  Rho4L1-2 Target-site Amplified by PCR: (rhoPCR1F:CAGTTGGGATCACAGGCTTC, rhoPCR1R:CAGGATGTAGTTTAGGGGTG) TAAGGATCCTTTGGGCAAAAAAGAAACAGAGAAGGCATTCTTTCTATACAAGAAAGGACTTGATAGAGCTGCTACCATGAACGGAACAGAGGGTCCCAATTTTTATATCCCCATGTCCAACAAAACTGGGGTGGTACGAAGCCCATTCGATTACCCTCAGTATTACTTAGCAGAGCCATGGCAATATTCAGCACTGGCTGCTTACATGTTCCTGCTCATCCTGCTTGGGTTACCAATCAACTTCATGACCTTGTTTGTTACCATCCAGCACAAGAAACTCAGAA  Rho4S1-1 Target-site Amplified by PCR: (4Snew:CACAGAAGGCATTCTTTCTAG, 4S-rhoPCR1R:CAGCAAGATGTAGTTTAAGGGTG) CACAGAAGGCATTCTTTCTAGATAGGAAAGGACTTTTTAGAGCTGCTACCATGAACGGAACAGAAGGTCCAAATTTTTATGTCCCCATGTCCAACAAAACTGGGGTGGTGCGAAGCCCCTTTGATTACCCTCAGTATTACTTAGCAGAGCCATGGAAATATTCAGCACTGGCAGCTTACATGTTCCTGCTCATCCTGCTTGGGTTTCCAATCAACTTCATGACCTTGTATGTCACCATCCAGCACAAGAAACTCAGAACACCCTTAAACTACATCTTGCTG  Rho4S1-2 Target-site Amplified by PCR: (4Snew1-2: CACAGAAGGCATTCTTTCTAT, 4S-rhoPCR1R:CAGCAAGATGTAGTTTAAGGGTG) CACAGAAGGCATTCTTTCTATACAAGAAAGGACTTGATAGAGCTGCTACCATGAACGGAACAGAGGGTCCCAATTTTTATATCCCCATGTCCAACAAAACTGGGGTGGTACGAAGCCCATTCGATTACCCTCAGTATTACTTAGCAGAGCCATGGCAATATTCAGCACTGGCTGCTTACATGTTCCTGCTCATCCTGCTTGGGTTACCAATCAACTTCATGACCTTGTTTGTTACCATCCAGCACAAGAAACTCAGAACACCCTTAAACTACATCTTGCTG  Exon 5 TTCCGTAACTGCTTGATCACCACCCTGTGCTGTGGAAAGAATCCATTCGGTGATGAAGATGGCTCCTCTGCAGCCACCTCCAAGACAGAAGCTTCTTCTGTCTCTTCCAGCCAGGTGTCTCCTGCATAA 	57		APPENDIX B Cloning sgRNAs into pDR274 for downstream in vitro transcription Linearize pDR274 with BsaI   1. Hybridize sgRNAs 1uL sgRNA “A”  1uL sgRNA “B” 8uL dH2O FV = 10uL Boil 7min  Cool at RT 30min Ice 10 min Dilute 1/11 (1uL in 10uL dH2O)   2. Ligation Experimental ligation reaction: 16uL dH2O 2uL 10X T4 buffer 0.5uL linear pDR274 (Vector) 1uL of sgRNA hybrids dilution (Insert)  Control ligation: 17uL dH2O 2uL 10X T4 Buffer 0.5uL linear pDR274 0.5uL T4 ligase Incubate @RT 1hr OR overnight  3. Transformation and plate on LB+KAN DAY 2 4. Set up liquid overnights  Pick four colonies on the experimental plate, inoculate 3mL LB+KAN of each colony Shake overnight @ 37°C, 225 RPM  DAY 3  5. Miniprep & Sequence Miniprep & Quantify with nanodrop  Send for sequencing according to CMMT requirements             	58		APPENDIX C Full Protocol for RNA Injection Experiments  IN VITRO TRANSCRIPTION OF RNAS Cas9 Linearize ~5ug of pMLM3613 with PmeI  DNA (Phenol-Chloroform?) Ethanol Precipitation - Resuspend in 8.7uL nuclease-free H2O - Nanodrop 0.5uL for quantification & purity  HiScribe ARCA kit - NEB: 1. Capped Assembly with ~1.5-2ug template DNA, ~1hr incubation 2. DNase digestion of template DNA, ~15min incubation 3. A-Tailing, ~40min incubation Column Clean-up RNeasy – Qiagen kit - (Ethanol Precipitation if combining 2 preps -nuclease-free Ammonium Acetate) - Resuspend in 21uL nuclease-free H2O - Confirm individual preps with nanodrop & gel before ethanol precipitation Aliquot 1uL in nuclease-free tube for gel & nanodrop Store stock at -80°C immediately Confirmation of successful in vitro transcription - Nanodrop for quantification & purity, use 0.5uL - Gel to confirm band size, no degradation, load with remaining 0.5uL  sgRNA – performed with two preps in parallel, combine preps in EtOH Precipitation Linearize ~5ug of pDR274 with desired sgRNA cloned-in with DraI DNA (Phenol-Chloroform?) Ethanol Precipitation - Resuspend in 12.7uL nuclease-free H2O - Nanodrop 0.5uL for quantification & purity MAXIscript Kit - Ambion: set-up 2 individual reactions 1. Enzyme Mix with ~1.5-2ug template DNA, ~1.5hr incubation 2. DNase digestion, ~15min incubation Column Clean-up miRNeasy – Qiagen Kit Aliquot 1.5uL, Confirm successful in vitro transcription: - Nanodrop for quantification & purity, use 0.5uL - Gel to confirm band size, no degradation, load with remaining 1uL Combined preps in Ethanol Precipitation best yield if left overnight at -20°C - Resuspend in 21uL nuclease-free H2O  Aliquot 1uL in nuclease-free tube for gel & nanodrop Store newly synthesized RNA at -80°C immediately Confirm ethanol precipitation with aliquoted 1uL: - Nanodrop for quantification & purity, use 0.5uL - Gel to confirm band size, no degradation, load with remaining 0.5uL  PREPARATION FOR MICROINJECTION EXPERIMENT Week Prior Finish in vitro transcription of Cas9 mRNA and appropriate sgRNA Determine injection schedule & reaction calculations Prepare Ficoll solutions (0.1XMMR + 6% Ficoll; 0.4XMMR + 6% Ficoll) Prepare Needles: 	59		- 20-25um diameter (best is 1 notch; not less than, not more than 1.5 notches) - 2X perfect needles/reaction + extras   -2 Days  Pre-priming injection of 50-100uL HCG in afternoon (~1400-1600), transfer females into 50mM salt bin  -1 Day Set injection room temperature to 18°C (before 1900?)  Prepare 2% agarose injection plates Prepare 0.1XMMR & 1XMMR  Store ficoll, MMR solutions, & injection plates in 18°C incubator Prime females with injection of 700uL HCG ~2300, transfer females to 20mM salt bin Prepare room: - Absorbent pads for under microscopes - 2 dissecting microscopes - A micromanipulator beside each microscope - Optics box (usually stays in there)  - Trolley with blue bin o Blue bin with 10mM NaCl - Row of petri dishes - Check that pump turns on Prepare RNA injection reactions  Spin stocks 13K, 2min, RT  Pipette off the top  INJECTION DAY Check that temperature of room is 18°C Check that females are laying eggs Needles, solutions, sharpies, timers, glass pipettes, waste beakers, pipettes & pipette tips in room  Plastic pipettes: 2 for loading, 1 for washing, 1 for cysteine, and 1 per in vitro fertilization reaction  Keep door closed for as long as you can once you have put everything in the room  Make cysteine (2%, pH 8.00, 1XMMR) Make 0.1% Tricane, 0.1X MMR (1.5mL 20XMMR, 300mg Tricane) Anesthetize male in 0.1% Tricane, 0.1XMMR for >20min  Take picture with voice recording  Confirm anesthetized state by flipping upside-down for >5min  Dissect out testes, store in 1XMMR (1mL in eppendorf)  Label empty eppendorfs for in vitro fertilization reactions, store in rack in injection room Set pump according to pump directions, prime pump lines Carry bin with females into room, put them into 10mM NaCl Blue Bin, keep other bin below Load 2 needles with 3uL RNA, back-prime with mineral oil - Switch to second needle when first needle runs out approximately halfway through  D0: INJECTIONS Squeeze females for embryos into empty petri dish Follow protocol for in vitro fertilization reaction While in vitro incubating, prepare injection plates  The next steps should be carried out as quickly as possible to prevent half-GFP+ embryos 	60		Dejelly with cysteine, wash with 0.1XMMR Label injection plates (on bottom) and fill with 0.4XMMR + 6% Ficoll Load plates with embryos Prepare monolayer  Spin RNA reactions 13K, 1min, RT Pipetting off the top, load 3uL RNA reaction, leave small air bubble, back-prime with mineral oil Proceed with injections (36uL/hr, diameter 1.03 = 10nL/embryo)  You will only finish half of the larger ½ plates with 3uL When first needle runs out, load 1.5uL in second needle and continue Transfer injection plates to 18°C cyclic light incubator In ~2.5hrs, transfer healthy four-cell embryos to 0.1XMMR + 6% Ficoll + 50ug/mL Gentamycin Transfer ficoll plates back to 18°C cyclic light incubator, leave overnight  D1 POST FERTILIZATION Transfer embryos to 0.1XMMR, remove dead, count how many alive/dead Screen for GFP, transfer GFP+ to separate petri dish - Transfer dysmorphics/dying to individual plate, screen dysmorphics for GFP - Genomic prep ~5 GFP+ dysmorphics, send for sequencing Send ~5 dysmorphic GFP+ embryos for sequencing (genomic prep, PCR, ExoSAP)  D2 POST FERTILIZATION Screen for GFP, transfer GFP+ to 0.1XMMR bin, keep GFP- plates until D3 Remove dead, count how many alive/dead  D3 POST FERTILIZATION Screen for GFP, transfer GFP+ to 0.1XMMR bin, euthanize remaining GFP+ embryos  D14 POST FERTILIZATION Sac & solubilize animals, ideally those with eyes >4 apart at 1.25X  Genomic prep tail tissue, Transfer body to paraformaldehyde   ANALYSIS Dot Blot for rhodopsin quantification Immunohistochemistry for phenotype Direct & Discrete Sequencing  SEQUENCING ANALYSIS PROTOCOL Genomic prep embryos/tail tissue Direct sequencing: PCR amplify target-sites (refer to Appendix A for primer sequences) Run agarose gel for confirmation of target-site amplification ExoSAP cleanup Sequencing reaction: 2uL ExoSAP’d PCR product, 1uL 10uM primer, 12uL ROH2O Graph trace reads using peak reporter, choose best candidates for discrete sequencing Discrete sequencing:  PCR amplify Rho4L rhodopsin (GA-rhoPCR1 primers) and Chr4S (GA-4SrhoPCR1new primers) Gel purify band  Gibson assembly:   4uL GAMM   0.22uL linear BS-SKII+ (linearized with EcoRV) = ~10ng 	61		  0.98uL gel purified PCR product    Incubate 50°C, 60min   4°C hold  Transformation    20uL competent cells + 5uL gibson assembly reaction   incubate on ice 10min   heat shock 30s at 42°C   incubate on ice 5min   Add 500uL LB   Shake 225 RRPM, 30min at 37°C   Plate 100uL on LB+AMP plates   Overnight in 37°C incubator  Pick 3-5 colonies, miniprep, sequence with -21 M13F universal primer   Analyze for indel frequency/character                                    	62		APPENDIX D Excision of RHO by co-injection of rhosg3 and rhosg4 It has been reported that there can be excision of a gene using an upstream and downstream targeting sgRNA. A fourth sgRNA, targeting the last exon of rhodopsin was designed, rhosg4. Embryos were injected with equal amounts of rhosg3 and rhosg4. Primers were designed so that if there was simultaneously dsDNA cleavage that resulted in the excision of the rhodopsin gene, an 200bp amplicon could be amplified by PCR. There were lower levels of rhodopsin in these animals, but all attempts to PCR-amplify the region from upstream the rhosg3 predicted cut-site to downstream the rhosg4 predicted cut-site were unsuccessful.                                          	63		APPENDIX E eGFP-HDR Construct Design  The below sequence was cloned into BS-SKII+. Rhodopsin cds is encoded as homology with the sequence for eGFP inserted at a proven position in the amino acid sequence. The rhosg4 target-site has been mutated to encode 2 silent mutations that prevent subsequent cleavage.  CCACCAGTCTACAGCTGTCATCTAGGAATGGTGGAAGTTTCAGTTCAACCAAAGCAAAGAGTGCTAAACATGAGCTGATTAACTATGACTCCCATTGTCTATAGCTGCAGTTAAAAAAAGTCATGGAGAATGTTCTGCTACTTATGATGACTGTGTTGCCTTAGGATCAGCATACAACTTGGGGTAGTAAGGGCATTTGTCTTAGGTCCCAAAATGGAAGGGGCTTTTAGAGGAGAAACACAGATAAAACTACAAAGACCACCAGTGTTTTAGTATTTTAAAACACGGGTAATAACCCTTGAGCAAACTTCGTGTGCTGGATTAATTATTACGTACCTTTCGTTGGGGGTGCCGTCCTCCTATGATCCATGCACCGTGAACGTTGAAAGAGGGACCAGGTGTGCATCTGCATTTCGTTTGGCTTAGTATTTTACAACCTTCGTATACATCAATATGCAATGCATTACTTATTTTTAGGGTAGTAGAGACTAAATGAGTACAACAATACAGAAGAGATGAATGGACTAGATAATGACTGTTCTCCTTTCTCTCCCCAGTTCCGTAATTGTTTGATCACCACCCTGTGCTGTGGAAAGAATCCATTCGGTGATGAAGATGGCTCCTCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCAGCCACCTCCAAGACAGAAGCTTCTTCTGTCTCTTCCAGCCAGGTGTCTCCTGCATAAGAGCTTCACCAGGGCTGTCTCAGGGTCCGCTGCCTCACACAATTCCCATCACTTAAGCCCTGTCTACTTGTTGCGAAGGCAAAGAATTCCACAGTTTTAATATTTACCCCCATTCTGCCCAACCTTGGACACTGTAAGAGCTGACCCCATTACTGCTGGGAAGGCCCAAGCTTTGTTGCATTCTGATGTGATCCTTTCAGCAGAAAATGGGTGGATTCAATGAATTTCACCAAGGCTGTACATAACAATAACATTAGTCTGAAGGCACCTCCCACCCAGAGAATGCAACACTTATTTATCTCTGTCTTTTCTTGACATATTGATGCTGCTTCTATTCATGGTCACTAACAAAAAGTCCCATTTTACAATGCAACTGAAAGTAATGTATTTTTGTAATATAATAACATATTTCATGCAATCTCCTCTGCTTATTGGCAAGGTCTGATATAGTGAGGATAGACAGCCAGACCC  Xenopus RHO eGFP PAM rhosg4 insertion site stop codon fusion site  CC ACC CAG AAG GCT GAG AAA GAG GTC ACC AGA ATG GTT GTT ATC ATG GTC GTT TTC TTC CTG ATC TGT TGG    T   Q   K   A   E   K   E   V   T   R   M   V   V   I   M   V   V   F   F   L   I   C   W       234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256  GTG CCC TAT GCC TAT GTG GCA TTC TAC ATC TTC ACC CAC CAG GGC TCC GAC TTT GGC CCA GTC TTC ATG ACT V   P   Y   A   Y   V   A   F   Y   I   F   T   H   Q   G   S   D   F   G   P   V   F   M   T    257 258 259 260 261 262 263 264 265 266 267 279 280 281 282 283 284 283 284 285 286 287 288 289  GTC CCA GCT TTC TTT GCC AAG AGC TCT GCT ATC TAC AAT CCT GTC ATC TAC ATT GTT TTG AAC AAA CAG V   P   A   F   F   A   K   S   S   A   I   Y   N   P   V   I   Y   I   V   L   N   K   Q    290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312  TTC CGT AAC TGC TTG ATC ACC ACC CTG TGC TGT GGA AAG AAT CCA TTC GGT GAT GAA GAT GGC TCC TCT GCA F   R   N   C   L   I   T   T   L   C   C   G   K   N   P   F   G   D   E   D   G   S   S   A    313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336  GCC ACC TCC AAG ACA GAA GCT TCT TCT GTC TCT TCC AGC CAG GTG TCT CCT GCA TAA A   T   S   K   T   E   A   S   S   V   S   S   S   Q   V   S   P   A   -  337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354   WT target-site AAC AAA CAG TTC CGT AAC TGC TTG ATC ACC ACC CTG TGC TGT GGA AAG AAT N   K   Q   F   R   N   C   L   I   T   T   L   C   C   G   K   N     Mutant target-site to prevent subsequent removal AAC AAA CAG TTC CGT AAT TGT TTG ATC ACC ACC CTG TGC TGT GGA AAG AAT N   K   Q   F   R   N   C   L   I   T   T   L   C   C   G   K   N     Primers for quickchange mutagenesis F: AGTTCCGTAATTGTTTGATCACCACCCTGTGC R: TGGTGATCAAACAATTACGGAACTGGGGAGAG  Full Xenopus Rhodopsin 4L1-1 peptide sequence MNGTEGPNFYIPMSNKTGVVRSPFDYPQYYLAEPWQYSALAAYMFLLILLGLPINFMTLFVTIQHKKLRTPLNYILLNLVFANHFMVLCGFTVTMYTSMHGYFIFGQTGCYIEGFFATLGGEVALWSLVVLAVERYMVVCKPMANFRFGENHAIMGVAFTWIMALSCAAPPLFGWSRYIPEGMQCSCGVDYYTLKPEVNNESFVIYMFVVHFTIPLIVIFFCYGRLLCTVKEAAAQQQESATTQKAEKEVTRMVVIMVVFFLIC	64		WVPYAYVAFYIFTHQGSDFGPVFMTVPAFFAKSSAIYNPVIYIVLNKQFRNCLITTLCCGKNPFGDEDGSSAATSKTEASSVSSSQVSPA-   APPENDIX F M13F-HDR Construct Design The below sequence was cloned into BS-SKII+. The entire sequence is rhodopsin cds encoding the M13F epitope sequence and the rhosg1 target-site has been mutated to encode silent mutations to prevent subsequent cleavage.  GTGGCTTATGGGTTAAAAAGGTGCAACACAAACAAATAATCTATTATTTACACACTAGTCAAGACTGGTGCTCAGCTGTGGTTTGAAGATTCTAATTCAATGAACTAATGGTAACCAGGGCCGGATTTGTATTTCTGCAGCCCCTAGGCCATGCGGTCCTAACGTCTGTCCACGACGAGTCTTATTGCCATCCACCCGCAACTCCCGCAAGTGCAAATTTTGGAGCACTGGTGCTCTTCAGCAAGTGGCTGGGCGGCATGCCGTCCCTAAAAGTTCGCCGCCCTAGGCACAGGCCTTTGTGGCCTCTCCACAAATCCAAGCCTGATGGTAACTAAATGTAGAGGGAACTGAGTAAACCCCAAAAATGGCTGCCCTGGCTCCTACAATATGGAATTATCTCCTGTAGGTCAGACCTGGATTTCTTCCTGTCACTTTTAAATACACTTTCTTCTTGTGTGTTTAACAGAGAGAGAGATTGACAGGTGTAGACTTAATACGTTTAAGGGAAGCCAATTAACACTTTGCAATTTTAGCTTGGATTACAGTGATTAATAGTGCGCTAAATCCTTTGTTGCTGACGCTGGGGGTTGCAAGCTTACTCCAGGTGGGACTTTAAAAGGACGAGGGGACAGTGGGTCATACTGTAGAACAGCTTCAGTTGGGATCACAGGCTTCTAGGGATCCTTTGGGCAAAAAAGAAACACAGAAGGCATTCTTTCTATACAAGAAAGGACTTTATAGAGCTGCTACCATGAACGGAACAGAAGGTCCAAATTTTTATGTCCCCTTTTCCAACAAAACTGGGGTGGTACGAAGCCCATTCGATTACCCTCAGTATTACTTAGCAGAGCCATGGCAATATTCAGCACTGGCTGCTTACATGTTCCTGCTCATCCTGCTTGGGTTACCAATCAACTTCATGACCTTGTTTGTTACCATCCAGCACAAGAAACTCAGAACACCCCTAAACTACATCCTGCTGAACCTGGTATTTGCCAATCACTTCATGGTCCTGTGTGGGTTCACGGTGACAATGTACACCTCAATGCACGGCTACTTCATCTTTGGCCAAACTGGTTGCTACATTGAAGGCTTCTTTGCTACACTTGGTGGTAAGTTCCAATGGGCTTTCGTCACTGATATTGTTGTAGCAATAAATTCTTGGAAAGCTCGTAAGGGAACAAGCTACCAGGGAAAGGGTTATAGGGCTGAAAAGGAATATCAGTACTTTCTATGTTCTCCAGCAGAGTGTAGTGCATACCATGTTAGAGAAAGTTCAACATGTAATACTGTGAGGGGCCAAATTGCCTGGGTTGAGTCATGTTAACCTTTTGCCTTTTCCTGTTCTTTTTACTAACAGGTGAAGTGGCCCTCTGGTCACTGGTAGTATTGGCCGTTGAAAGATATATGGTGGTCTGCAAGCCCATGGCCAACTTCCGATTCGGGGAGAACCATGCTATTATGGGTGTAGCCTTCACATGGATCATGGCTTTGTCTTGTGCTGCTCCTCCTCTCTTCGGATGGTCCAGGTAAATATATATAACATCAGTCAGCATATCCCTAG  TTT – M13F M13F-F Start Codon M13F+ M13F+/- M13F- M13F-R rhosg1  WT target-site: V   V   R   S   P   F   D   Y   P   Q   Y   Y   L   A   E GTG GTA CGA AGC CCA TTC GAT TAC CCT CAG TAT TAC TTA GCA GAG CC  Mutant peptide sequence  V   V   R   S   P   F   D   Y   P   Q   Y   Y   L   A   E GTG GTA CGA AGC CCA TTC GAT TAC CCT CAA TAC TAC TTA GCA GAG CC  Quick-change paper indicated 24bp overlap preferable  Xia, Y., Chu, W., Qi, Q., & Xun, L. (2015). New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis. Nucleic Acids Research, 43(2), e12–e12. http://doi.org/10.1093/nar/gku1189  F: ATTACCCTCAATACTACTTAGCAGAGCCATGG R: CTGCTAAGTAGTATTGAGGGTAATCGAATGG  “The Phusion PCR was done in 20  l with 2 ng of pBS-TAA as template and 0.5  M partially overlapping primers in the Phusion HF buffer. The PCR was done with initial denaturation at 98◦ C for 3 min, followed by 16, 20 or 25 cycles of denaturizing at 98◦ C for 25 s, annealing at 69◦C for 30 s and extension at 72◦C for 90 s, and the final step was incubation at 72◦C for 10 min” - 20 PCR cycles preferable according to colony count data        	65		     APPENDIX G Cloning methods for sgRNAs into hU6 & xtU6 (Ligation & Gibson Assmbly)  Jinek et al. (2012). Science  Above is visual representation of oligo design orientations Oligos must be exactly adjacent to ‘NGG’ (PAM) 20bp of “non-complementary strand” exactly 5’ of PAM is verbatim target-site sequence  LIGATON METHOD 5’ phosphorylated oligos ligated into hU6 BbsI linearized, dephosphorylated, plasmid   Ran et al. (2013). Nature Protocols   Ran et al. (2013). Nature Protocols  Oligo A: CACC + G + target-site 20nt Oligo B: AAAC + reverse complement of target-site 20nt +C Oligos must be 5’ phosphorylated for cloning purposes 	66		Always consider orientation to PAM     CLONING PROTOCOL 1. Linearize plasmid with BbsI restriction digest; 37°C 1-3hrs   DNA   = ~1ug   BbsI    = 1U   NEB 2.1 = 5uL   ROH2O   = FV 50uL   2. Dephosphorylation with CIP   1uL CIP   60min @ 37°C  3. Gel extract band; run control for supercoiled DNA  4. Hybridize oligos   1uL A + 1uL B + 8uL ROH2O   Boiling water (98°C) 7min   RT 30min   Ice 10min  5. Ligation reaction    100ng Vector = x uL   540pg Insert = 1.82 uL of a 1/500 dilution of hybrid oligos   10X T4 ligase = 2uL   T4 Ligase = 0.5uL    ROH2O = FV 20uL  6. Transformation   1uL ligation in 4uL TE   Mix with 45uL comp cells   Vortex 10s on setting 3   30min ice   30s @ 42°C   10 min ice   500uL LB   1hr @ 37°C, shaking 225 RPM   Spin down cells, 8K RPM 2min   Remove 400uL supernatant   Plate remaining on appropriate selection plate  7. Miniprep & Sequence       	67		     GIBSON ASSEMBLY METHOD Gibson Assembly reaction for cloning into BbsI linearized  Blue = hU6 promoter region Light Blue = BbsI restriction sites Pink = chimeric sgRNA BB  Light Blue = Now with target-site sequence Purple = +1G transcription start site  Annealed/Extended 69mer Oligos To design the GA oligos, take this sequence, -14mer of +1G ATCTTGTGGAAAGGACGAAACACC + G + 20nt target-site sequence  +14mer of end of target-site in reverse complement ACTTGCTATTTCTAGCTCTAAAAC + RC target-site + C  PROTCOL:  1. Anneal/Extension 98…  2. GARXN  50ng 3. Transformation … 4. Miniprep, BbsI test digest, & Sequence  Hybridized 60mer Oligos  To design the GA oligos, take this sequence, -14mer of +1G GTGGAAAGGACGAAACACC + G + 20nt target-site + GTTTTAGAGCTAGAAATAGC … and the reverse complement + 19bp homology, - 20bp homology  PROTCOL:  1. Hybridize Oligos 98…  2. GARXN  50ng 3. Transformation 	68		… 4. Miniprep, BbsI test digest, & Sequence    APPENDIX H XOP-Cas9-GFP-xtU6  XOP0.8 Cas9-GFP xtU6 sgRNA Backbone BbsI  TAGTTATTACTAGCGGCCGGCCAGTTCGACGATGTAGGTCACGGTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCACCTCACCCATCTGGTCCATCATGATGAACGGGTCGAGGTGGCGGTAGTTGATCCCGGCGAACGCGCGGCGCACCGGGAAGCCCTCGCCCTCGAAACCGCTGGGCGCGGTGGTCACGGTGAGCACGGGACGACGTGGAGTTGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAGACAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGAGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCAACTCGACGTGCGACGGCGTCGGCGGGTGCGGATACGCGGGGCAGCGTCAGCGGGTTCTCGACGGTCACGGCGGGCATGACTGATATCGTCTATGGCCAAACAGAGCCCCCTCTGGGCTGCAAATCCACAAAGGGCTACCAAATAGACAATCATATTCTTTATTAGGCACCCCAAGGGCTTTTTTCACGCTTGTGCTGCTCCGCAACTCTTTTTACATTTGAATGTGGCTTATGGGTTAAAAAGGTGCAACACAAACAAATAATCTATTATTTACACACTAGTCAAGACTGGTGCTCAGCTGTGGTTTGAAGATTCTAATTCAATGAACTAATGGTAACCAGGGCCGGATTTGGATTTCTGCAGCCCCTAGGCCATGCGGTCCTAACGTCTGTCCACGACGAGTCTTATTGCCATCCACCCGCAACTCCCGCAAGTGCAAATTTTGGAGCACTGGTGCTCTTCAGCAAGTGGCTGGGCGGCATGCCGTCCCTAAAAGTTCGCCGCCCTAGGCACAGGCCTTTGTGGCCTCTCCACAAATCCAAGCCTGATGGTAACTAAATGTAGAGGGAACTGAGTAAACCCCAAAAATGGCTGCCCTGGCTCCTACAATATGGAATTATCTCCTGTAGGTCAGACCTGGATTTCTTCCTGTCACTTTTAAATACACTTTCTTCTTGTGTGTTTAACAGAGAGAGAGATTGACAGGTGTAGACTTAATACGTTTAAGGGAAGCCAATTAACACTTTGCAATTTTAGCTTGGATTACAGTGATTAATAGTGCGCTAAATCCTTTGTTGCTGACGCTGGGGGTTGCAAGCTTACTCCAGGTGGGACTTTAAAAGGACGAGGGGACAGTGGGTCATACTGTAGAACAGCTTCAGTTGGGATCACAGGCTTCTAGGATATCAGATCTCGAGCTCAAGCTTCGAATTCgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGNLS3X FLAGEcoRIXhoIXOP0.8EcoRVEcoRVBbsIEnhancerDislike-like element (inverted enhancer & enhancer)XbaINotIEcoRIpUC ori (temp)DI (tempp)pXOP Cas9-GFP xtU6sgBB11708 bphSpCsn1Cas9-GFP ORF (ORF3 1251-1611)FseIDI (temp)AflIIIEnhancer motif inverted!BbsIpoly(T) terminatorsgRNA backboneGFPNLSXenU6 PromoterXbaITATA boxBbsI cut-siteElement required for correct 3' formation of U6 matEcoRVNeo (temp)	69		CCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGgaattcGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGgaattctaaGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAgAAAGGCAAGCGCAAAAGGCCAACAACTGGCGCAACAAGCGAAGGAGAGGCCTTTAAAATGACAGGGAGCCGCGGCGGCAGGGCGGCGCTGTTTATTAAATGCTAGCGAGAGCACTGTGGGAAAATATGCATCGCCAAGTGGCGCGGCCAAAAAGCCCACGAGGGCGTGCGCGACTAAACTCTGGCAGCTCAAGCAAAAGGGAACTCCCCATTCTGCCGCCTGCGCTTATTTCCGCAAATCTAGGCGCGTTTAAGGGTCGGACGATTTGCGTCCAGCGAACGCTCAGCCTGCTTGGTAGGGCTATTGACATGGAGTCCAGGCAGTGCACAAACTCTTTGCCTCTGGTCTTCCCAGTTTGCTAAACTGCTAGGTGGAGGTAGGTAGGCGTGGCGTCAGCCCCACGGATCGTCTAGGCAGGTAGGAGAGAATAAAAGGTCATTTGCATATGCAAACGCCCTGCCCGTGTGGCAGATGATTGTTACCTAAAATATCAATTTCCCCTCTAGAAAACAGAGGTCCATGAAAATATTAAAAGGGGCTTTTGGGCTAATGCTTCTTTAGTGCTACAAGCAGATGACATCTAAACTTTGAAATTGGCACTTTGGTTCCATCTGTCACTCTCCTTAAGTTACAGGGGTTCCATCTGGCGGCGCTTATAAGGCGGTGCTGCTCGCTGGAGTTTCCACCggGTCTTCgaGAAGACctgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTAAGCTCCTCGGCAAAATCAAAGAAGTTATCCACGGAGCAGAAGCATCCCTGCAATAGCAAAAGTGGAGAAATAAGGCAGGCGGAAGAAATGAAAGCGCTCACATGAGCTCCTAGAATGGCCCGACGCCATGTTTTGTCACGCGAGGGAAAGGAGCAAGCGAGAGGGctgcagacGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC	70		CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTGAGTTGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAGACAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGAGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCAACTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT         APPENDIX I CMV-Cas9-GFP-hU6   XOP0.8 Cas9-GFP hU6 sgRNA Backbone BbsI  TAGTTATTACTAGCGGCCGGCCAGTTCGACGATGTAGGTCACGGTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCACCTCACCCATCTGGTCCATCATGATGAACGGGTCGAGGTGGCGGTAGTTGATCCCGGCGAACGCGCGGCGCACCGGGAAGCCCTCGCCCTCGAAACCGCTGGGCGCGGTGGTCACGGTGAGCACGGGACGACGTGGAGTTGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAGACAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGAGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCAACTCGACGTGCGACGGCGTCGGCGGGTGCGGATACGCGGGGCAGCGTCAGCGGGTTCTCGACGGTCACGGCGGGCATGACTCTTAAGTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGNLS3X FLAGEcoRIXhoIMegaprimer RCMV promoterMegaprimer FDI (temp)FseIEcoRVhSpCsn1Cas9-GFP ORF (ORF3 1251-1611)KflINeo (temp)extraU6 TerminatorsgRNA backboneBbsIBbsITATA boxU6 promoterAflIIISV40 poly(A) signalNotIGFPEcoRINLSNLSpUC ori (temp)pCMV Cas9-GFP hU6sgBB10959 bpAflIIDI (tempp)	71		TATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCAGGCCAGGCAAAAAAGAAAAAGgaattcGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGgaattctaaGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCggGTCTTCgaGAAGACctgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTgttttagagctagaaatagcaagttaaaataaggctagtccgtTTTTagcgcgtgcgccaattctgcagacTTAAGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGT	72		GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTGAGTTGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAGACAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGAGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCTCCCAGGCGCGCAACTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT        APPENDIX J Example of direct sequencing base call analysis.   WT PRIMARY SECONDARY  VALUE: A A  G   1 G A  G   0.25 R A  G   1 R A  T   0.5 T A  G   0  	73		 http://www.chem.qmul.ac.uk/iubmb/misc/naseq.html         	74		APPENDIX K WT Alignments for section 2.7           	75		   D1 Embryos: WT-2 = 4L homeolog; 4SWT-2 = 4S homeolog; rhosgN = 4L homeolog    	76		APPENDIX L NLS Change in Cas9-GFP  FseI site in NLS  Proposed bp change  NLS predicted peptide sequence with expasy: KRPAATKKAGQAKKKK   AAA AGG CCG GCG GCC ACG AAA AAG GCC GGC CAG GCA AAA AAG AAA AAG K   R   P   A   A   T   K   K   A   G   Q   A   K   K   K   K  AAA AGG CCG GCG GCC ACG AAA AAG GCA GGC CAG GCA AAA AAG AAA AAG K   R   P   A   A   T   K   K   A   G   Q   A   K   K   K   K   Cas9 NLS FseI T2A GFP SanDI Oligos   …CCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCAGGCCAGGCAAAAAAGAAAAAGgaattcGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA…  Oligos ordered: GCGATAAGCTGATCGCCAGAAAGAAGGACTGG AATTCCTTTTTCTTTTTTGCCTGGCCTGCCTTTTTCG 	77		APPENDIX M xtU6 design error    The “G” of the desired oligo is actually now -28 away from the TATA, instead of -23.   

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